HIV vaccines based on Env of multiple clades of HIV

ABSTRACT

The invention provides a composition comprising a pharmaceutically acceptable carrier and six plasmids, each of which encodes an HIV Env, Gag, Pol, or Nef protein. The invention also provides a method of inducing an immune response in an animal using the composition.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/818,113, filed Jun. 13, 2007, and issued as U.S. Pat. No. 7,666,427,which is a continuation of U.S. patent application Ser. No. 11/376,484,filed Mar. 15, 2006, and now abandoned, which is a continuation ofcopending International Patent Application No. PCT/US2004/030284, filedSep. 15, 2004, designating the U.S. and published in English on Apr. 21,2005 as WO 2005/034992, which claims the benefit of U.S. ProvisionalPatent Application No. 60/503,509 filed Sept. 15, 2003, all of which areexpressly incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is related to the field of vaccines against HIV.

2. Description of the Related Art

There is a need for a safe and effective vaccine against ever-mutatingHuman Immunodeficiency Virus (HIV). One requirement of a highlyeffective AIDS vaccine is the need to induce both neutralizingantibodies and cellular immunity to the many strains of HIV-1 thatcirculate throughout the world.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a multiclade HIV plasmid DNAor viral vector vaccine including components from different clades ofEnv (optionally Env chimeras) and Gag-Pol-(optionally)Nef from a singleGlade. The vaccine of the invention may further include V1, V2, V3, orV4 deletions or combinations thereof. In another embodiment, theinvention provides a multiclade HIV envelope immunogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of HIV Env vectors with V3 regionreplacements. A. The CXCR4-tropic HIV HXB2, a clade B gp140ΔCFI, wasmade as described previously (Chakrabarti, B. K. et al. 2002 J Virol76:5357-5368). Most divergent region including the V3 regions, from HIVHXB2 was replaced by the similar region of HIV BaL to make R5 tropicclade B HIV HXB/BaL. The gp140ΔCFI of both clade A and clade C were alsomade as described in the Materials and Methods (see PART I). B.Expression of the indicated vectors was confirmed by transfection in 293cells and Western blot analysis. The Env was detected by Western blotwith polyclonal antibody against gp160 (Intracel, Rockville, Md.) at adilution of 1:3000.

FIG. 2. Induction of neutralizing antibodies by chimeric Env with V3region substitutions. A. Neutralizing antibody activity from guinea pigsimmunized with HIV HXB2/BaL gp140ΔCFI. Immune sera were tested for theirability to inhibit HIV IIIB (open bars) and HIV MN (filled bars). Theneutralizing antibody titer is defined as the dilution of sera yielding50% virus neutralization in the MT2 assay killing (Montefiori, D. C. etal. 1988 J Clin Microbial 26:231-5). B. The same sera shown in FIG. 2Awere tested against HIV BaL. The data represent the % neutralization ofthe HIV BaL by these sera at 1:4 dilution.

FIG. 3. Titer and specificity of neutralizing antibodies generated inguinea pigs after immunization with gp145/140ΔCFI Envs. A. V3-specificneutralization of HIV BaL was measured in peripheral blood mononuclearcells (PBMC) using serum samples that were pre-incubated in the presenceand absence of different V3 peptides as described previously (Bures, R.et al. 2000 AIDS Res Hum Retroviruses 16:2019-35). Sera were tested at1:5 dilution in the PBMC assay. B. V3 peptide-specific neutralizingactivity induced by gp145/140ΔCFI of HIV BAL was detected by a reductionin the titer of HIV MN-specific neutralizing antibodies in the presenceof either HIV IIIB or HIV BAL V3 peptides compared to the untreatedcontrol. Assays were performed in MT2 cells as described in Materialsand Methods section of PART I (Montefiori, D. C. et al. 1988 J ClinMicrobial 26:231-5). The dashed line corresponds to a 50% cut-offconsidered positive for neutralization.

FIG. 4. Schematic representation and expression of different 2F5/V3mutations in HIV HXB/BaL ΔCFI Envs. A. Schematic representation ofgp145ΔCFI derived from clade B HIV HXB/BaL with 2F5 epitopes expressedin V3. Functional domains and major structural motifs are indicated, aspreviously described (Chakrabarti, B. K. et al. 2002 J Virol76:5357-5368). V1, V2, V3, and V4 refer to the respective variableregions, and the sequences of the relevant V3 loops are shown. Heptadrepeat-2 (HR-2), the coiled-coil peptide sequence upstream of thetransmembrane domain in R5/clade B envelope, was replaced by the similarregion from the clade C Env. The nucleotide sequence corresponding tothe amino acids at the tip of V3 (GPGRA, SEQ ID NO: 12) was replaced bynucleotide sequences corresponding to the polypeptide containing eitherthe minimal 2F5 epitope or by nucleotide sequences corresponding to thepolypeptide containing the extended 2F5 epitope.CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC (SEQ ID NO: 1);CTRPNNNTRKAIHIFYTTGEIIGDIRQAHC (SEQ ID NO: 2); LELDKWAS (SEQ ID NO: 3);KNEQELLELDKWAS (SEQ ID NO: 4); KNEKDLLALDSWRN (SEQ ID NO: 5). B.Expression of 2F5 mutant gp140ΔCFI envelopes. Expression of theindicated gp140ΔCFI B(C-HR2) or −2F5, gp140ΔCFIAGPGRA B(C-HR2) or -tip−2F5, gp140ΔCFI B(C-HR2) ext 2F5, V3 ext 2F5, and clade B gp140ΔCFI areshown. The indicated proteins were detected by immunoblotting asdescribed in Materials and Methods section of PART I. Cell-freesupernatants produced by transfection with vector containing no insertwere used as controls. C(C-1, C-2). Analysis of the reactivity of 2F5modified Env with monoclonal antibody, 2F5, and HIV-1 IgG. Binding ofgp140ΔCFI indicated mutants or controls transfected with vector alonewere analyzed by ELISA with monoclonal antibody 2F5 (left panel) andHIV-1 IgG (right panel). The values represent the mean and standarddeviation (error bars) for each point.

FIG. 5. Antibody response to 2F5 peptide in immunized guinea pigs.Comparison of the antibody response by ELISA in guinea pigs immunizedwith designated expression vectors. Sera collected 2 weeks after DNA(A.) and ADV boosting (B.) were used to detect the antibody that couldbind with 2F5 peptide. Serum from an animal immunized with the controlvector alone served as a negative control. C(C-1, C-2, C-3). Percentneutralization of the 2F5/V3 mutants in HIV HXB/BaL ΔCFI Envs is shownagainst a panel of HIV-1 clade B strains at 1:5 antibody titer. Fourindividual sera from 2F51V3 mutants immunized guinea pigs were screenedagainst HIV BaL, HIV IIIB, and HIV SF162 viruses. Percent neutralization(compared with corresponding pre-immune sera) is indicated.

FIG. 6. Interaction of gp140ΔCFI with different monoclonal antibodies orCD4 of gp140ΔCFI from different Glades. A. Analysis of the antigenicstructure of soluble gp140ΔCFI with monoclonal antibodies. Envglycoproteins from the supernatants of 293 cells transfected with theindicated vector expressing gp140ΔCFI were immunoprecipitated witheither monoclonal antibodies (5 μg) 2F5, 2G12, F105, and IgG1b12 or with5 μg of HIV-1 IgG. The proteins were analyzed by SDS-PAGE and detectedby Western blotting using the IgG from the pooled sera of the patient(HIV-1 IgG). The bands that cross-reacted with the antibody arepresented. B. Interaction of soluble gp140ΔCFI protein with CD4. Bindingof gp140ΔCFI and gp160, compared to that of controls transfected withvector alone, in an ELISA with CD4 is shown. The values represent themean and standard deviation (error bars) for each point.

FIG. 7. Comparison of breadth and potency of the neutralizing antibodyresponse induced by ΔCFI envelope from clade B (HXB/BaL) and from acombination of clades A, B, and C. A, B. Neutralization assays ofindicated viruses at a 1:5 dilution of four individual guinea pig sera.The percent neutralization was calculated by direct comparison of immunesera to the corresponding animals pre-immune sera. The single-roundintracellular p24-antigen flow cytometric HIV-1 neutralization assay hasbeen described previously (Mascola, J. R. et al. 2002 J Virol76:4810-21). Panel A shows the results from four guinea pigs immunizedwith the clade B Env immunogen. Panel B shows results from four guineapigs immunized with the multiclade immunogen. All sera were evaluatedagainst a panel of 19 viruses (shown on X-axis). Due to the large numberof viruses evaluated, data shown are from a single experiment for eachsera and virus. Comparison of neutralization by monoclade to multicladesera for any given virus (DJ263, ZA12, TV1 and DU151) revealed asignificant difference (p=0.029). C(C-1, C-2). V3 peptide competitionanalysis of the neutralization of clade B HIV 89.6 and BR07.Neutralization of HIV 89.6 (left) and HIV BR07 (right) by sera fromguinea pigs immunized with HIV HXB/BaL immunogen was tested at 1:5dilution. The serum was incubated with no peptide (mock), or 20 μg/ml of23mer V3 peptide based on the HIV BaL sequence (BaL V3) or an unrelatedmixture of peptides derived from Ebola GP (Ebola). Infection by HIV 89.6and HIV BR07 was completely inhibited by the HIV BaL V3 peptide but notby the control peptides. Data from a representative guinea pig serum isshown. D, E. V3 peptide competition analysis of the neutralization ofclade B HIV SF162. Panel D shows sera from two representative guineapigs; one immunized with the clade B Env immunogen (monoclade) and oneimmunized with the clade A, B, C Env immunogen (multiclade). Sera weretested at a 1:5 dilution and incubated with increasing concentrations ofthe 23mer V3 peptide based on the HIV BaL sequence. Note thatneutralization by the monoclade sera, but not the multiclade sera, wascompletely inhibited by the HIV BaL V3 peptide. The control values showthat the scrambled V3 peptide had no effect on serum neutralization. Thebar graph E displays data from neutralization of the HIV SF162 by thesame multiclade guinea pig serum, also at a 1:5 dilution. The serum wasincubated with 20 μg/ml of either the clade A, B or C V3 peptide, orwith 60 μg/ml of a combination of all three peptides (panel E). Acombination of all three V3 peptides did not reverse the majority of theserum-mediated neutralization of SF162. Error bars are the mean (+/−SEM)of two independent experiments. Both experiments shown in panel C weredone with a single serum, but all four sera in each group (monoclade ormulticlade) gave similar results.

FIG. 8. Comparison of immune response of multivalent multi-plasmids withsingle gene approaches. Four groups of mice with 5 mice per group wereimmunized with the control vector alone (50 μg), Env (25 μg) withcontrol vector (25 μg) as filler DNA, Gag-Pol-Nef (25 μg) with controlvector (25 μg) as filler DNA, or Env (25 μg) with Gag-Pol-Nef (25 μg).Ten days after the final immunization, splenic cells were harvested andsensitized with B-env peptide pool (158-peptide pool of Clade B Envprotein) and B-gag peptide pool (122-peptide pool of Clade B Gagprotein). Six hours later, the cells were fixed, stained with monoclonalantibodies, and analyzed by FACS to detect the IFN-γ and TNF-α positivecells in the CD4 (top row) and CD8 positive (bottom row) populationshown in FIG. 8A (A-1, A-2, A-3, A-4). In the FIG. 8B, mouse sera werecollected to detect antibody against Env using ELISA. ELISA plates wereprepared and coated as described in Materials and Methods section ofPART II with supernatant from cells transfected with pVRC2801 (R5gp140ΔCHI-Clade-B) from Clade B. Mouse sera from different groups werediluted starting from 1:100 to 1:2700 before testing. The ELISA titersare shown for the group immunized with pVR1012 (▴), withpVR1012-B-Gag-Pol-Nef and filler DNA (▪), with pVR1012-B-gp 145ΔCFI andfiller DNA(●), or with 1012-B-gp145ΔCFI+1012-B-Gag-Pol-Nef (♦). Eachpoint represents the average OD reading from the five animals per group.

FIG. 9. T cell and antibody responses in mice immunized Gag-Pol-Nef andclade B Env compared to Gag-Pol-Nef and clade A, B, C Env proteins. Mice(n=3) were immunized with a total of 50 μg of control vector,Gag-Pol-Nef and clade B Env (1:1 ratio), or Gag-Pol-Nef and Env fromclades A, B, and C (1:0.33:0.33:0.33 ratio). (A). Ten days after thefinal immunization, splenic cells were harvested and sensitized with aB-Env peptide pool (158 peptide pool of Clade B Env protein). Forcontrols, Ebola glycoprotein peptide pool (22 peptides) or unstimulatedcells served as a negative controls, and PMA was used as the positivecontrol. Six hours later, the cells were fixed, stained with monoclonalantibodies, and analyzed by FACS to detect the IFN-γ and TNF-α positivecells in the CD4 (left panel, 9A-1) and CD8 (right panel, 9A-2) positivepopulations. The symbols depict the individual results for the ten micein each group. The thin horizontal bar represents the average of the tendata points with a standard deviation error bar. B (B-1, B-2, B-3). Serafrom the three groups of animals were collected 10 days after the thirdimmunization, and ELISA was performed to detect the antibody against therespective clade Env's as described in Materials and Methods section ofPART II. Mouse sera from different groups were diluted from 1:200 to1:800 for testing. Each bar represents the average OD reading from thethree mice per group.

FIG. 10 (A-1, A-2, A-3, B-1, B-2, C-1, C-2, C-3). CD8+T cell responsesto different clade and gene combination vaccine candidates byintracellular cytokine analysis. Three groups of mice were immunizedwith a control vector (VR1012), ABC (×4) or ABC (×6) as described inTable 1. Ten days after the final immunization, splenic cells wereharvested and sensitized with the following peptide pools: A-Gag (125peptides), B-Gag (122 peptides), C-Gag (105 peptides), A-Env (154peptides), B-Env (158 peptides), C-Env (154 peptides), B-Pol-1 (120peptides from the first half of Clade B Pol), or B-Pol-2 (128 peptidesfrom the second half of clade B Pol). Cells were stimulated and analyzedby FACS, with positive and negative controls as in the legend to FIG. 9to detect the IFN-γ and TNF-α positive cells in the CD8+ population. Thesymbols show the individual results for the ten mice in each group. Thethin horizontal bar is the average of the ten data points with standarddeviation bars.

FIG. 11. CD4+T cell and antibody responses to combination gene and Gladevaccine candidates by intracellular flow cytometry and ELISA. Threegroups of mice were immunized with the indicated control or combinationvaccines as shown in Table 1. (A). Ten days after the finalimmunization, splenic cells were harvested and sensitized with theindicated peptide pools as described in the legend to FIG. 9. Individualresponses are shown with the symbols, and the thin horizontal bardepicts the average of the ten data points with a standard deviationerror bar. B (B-1, B-2, B-3). Sera from the three groups of animals werecollected 10 days after the third immunization, and ELISA was performedto detect the antibody against envelope as described in Materials andMethods section of PART II. Mouse sera from different groups werediluted starting from 1:100 to 1:2,700 for testing. Each bar representsthe average OD reading from the ten mice per group.

FIG. 12. Schematic representation of Envelope mutations. A. The majorstructural motifs in HIV Env are shown, together with the selectedexpression vectors used in these studies. V1, V2, V3, and V4 indicatethe respective variable regions and the sequence of the relevant V3loops are indicated (SEQ ID NO: 1). B. Schematic structure of the V3loop and V3 (1AB) stem-shortening mutations are indicated (SEQ ID NO:1).

FIG. 13. Mutation in the stem of the V3 loop and protein expression ofvarious gp145ΔCFI (HXB2/BaL chimera) V3 deletion mutants. A. Sequencesof progressive V3 stem deletion mutations in Env from HXB2/BaL chimera.B. Protein expression of gp145ΔCFI (HXB2/BaL chimera) V3 deletion mutantexpression vectors. The indicated mutations in the gp145ΔCFI constructs,described previously (Chakrabarti, B. K. et al. 2002 J Virol76:5357-5368), were prepared and analyzed by SDS-PAGE followed byWestern blot analysis with human monoclonal antibody 2F5. Plasmidexpression vectors encoding the indicated mutants were transfected into293 cells by use of calcium phosphate. Cell lysates were collected 48hours later. C. Expression of gp145 (HXB2/BaL chimera) V3 deletionmutant vectors with the V1 and V2 regions deleted.

FIG. 14. Effects of mutations in the stem of the V3 loop on tropism ofthe 89.6P Env. A, B (B-1, B-2, B-3, B-4, B-5, B-6, B-7). Buoyant densitysedimentation analysis of indicated V3 mutants in lentiviral vectorparticles, performed as described in Materials and Methods section ofPART III. C(C-1, C-2). Effects of V3 mutations in strain 89.6P Env oninfection of a CXCR4-tropic cell line, MT-2 (left), and a CCR-5 tropicindicator cell line, MAGI-CCR5 (right), using a liciferase reportergene. The positions of the indicated V3 mutations in strain 89.6P Envare the same as shown for the HXB2/BaL chimera (FIG. 13A). Bothcodon-modified and wild-type (wt) 89.6P Envs were used as positivecontrols.

FIG. 15. Expression of different HIV gp145 (HXB2/BaL chimera) V regionmutants and induction of neutralizing antibodies. A. Expression of theindicated HXB2/Bal V region mutants was determined by SDS-PAGE followedby Western blotting in transfected 293 cells. B. Neutralizing activityagainst BaL in sera from guinea pigs immunized with the indicatedDNA/ADV expression vectors. Sera were tested at 1:5 dilution. Resultsare the mean (+/−SD) for four guinea pig sera for each construct. The Pvalue shown is the result of a Mann-Whitney test comparing the mediumneutralization value of the two groups indicated.

FIG. 16. Characterization of antibody response induced by gp145(HXB2/BaL chimera) AV and selected V3 region mutants. A. Neutralizationactivity against BaL induced by immunization of guinea pigs with theindicated mutants, including the ΔV1V2 deletion mutants and theΔV1V2V3(1AB) stem-shortening mutants. Sera were tested at 1:5 dilution.Results are the means (+/−SD) for four guinea pig sera for eachconstruct. Results from one of two independent experiments are shown.The sera tested were independent from the sera tested for FIG. 15B. B.Total ELISA titers in the same guinea pig sera are shown and arecomparable between the different mutants.

FIG. 17. Comparison of breadth and potency of the antibody responseinduced by selected V region mutants. A (A-1, A-2, A-3, A-4). IC₅₀titers against four Glade B primary isolates are indicated (seeMaterials and Methods section in PART III). Results are themeans±standard errors of four guinea pig sera for each construct. Thestatistically significant differences between ΔV1V2V3(1AB) and eitherthe wild-type gp140/145 or gp140/145ΔCFI are shown (Mann-Whitney test).B. Four individual sera from ΔV1V2V3(1AB) immunized guinea pigs werescreened against a panel of 10 primary viruses. Sera were tested at a1:5 dilution. Percentages of neutralization (compared with correspondingpre-immune sera) are indicated. Data shown are an average of twoexperiments.

FIG. 18. In vitro expression of HXB2/Bal and 89.6P Env by both plasmidsand rADV vaccine constructs. The plasmid Env [gp145ΔCFI(R5) andgp145ΔCFI(89.6P)] and rADV [ADV-gp140ΔCFI(R5) and ADV-gp140ΔCFI(89.6P)]vaccine constructs were expressed in vitro, and protein expression wasassessed by Western blotting with human anti-HIV IgG.

FIG. 19 (A-D). Vaccine-elicited PBMC IFN-γ ELISPOT responses to SIVmacGag-Pol-Nef and HIV-1 Env. Freshly isolated PBMCs were assessed forIFN-γ ELISPOT responses after in vitro exposure to peptide poolsspanning the SIVmac Gag-Pol-Nef and HIV-1 Env proteins. All Env-specificresponses were assessed by using peptides that were matched to the Envimmunogen. The terms “matched” and “mismatched” refer to therelationship between the Env immunogen and the challenge virus. Arrowsindicate time of inoculation with either DNA or rADV immunogens. Dataare presented as the total antigen-specific SFC responses to Gag-Pol-Nefand HIV-1 Env per 10⁶ PBMCs and represent the mean values for sixmonkeys±standard error.

FIGS. 20 A and B. Vaccine-elicited PBMC IFN-γ ELISPOT responses toindividual viral proteins assessed 2 weeks following rADV boosting.ELISPOT responses to SIVmac239 Gag and Poi and HIV-1 Env antigens wereassessed. Env-specific responses were assessed with peptides that werematched to the Env immunogen, and mock Env-vaccinated monkeys wereassayed with 89.6P peptide pools. ELISPOT assays were performed on wholePBMCs (A) or PBMCs depleted of CD8⁺ T lymphocytes (B). Data arepresented as the mean SFC responses to individual viral proteins per 10⁶PBMCs and represent the mean values for six experimentally vaccinatedmonkeys±standard error.

FIG. 21. Postchallenge peripheral blood CD4⁺ T-lymphocyte counts. Thesevalues represent the mean percentage of CD3⁺ CD4⁺ lymphocytes assessedprospectively on all experimental monkeys through day 168 postchallenge.

FIG. 22. Postchallenge plasma viral RNA levels. These values weredetermined by an ultrasensitive bDNA amplification assay with adetection limit of 50 copies/ml. The values plotted represent thegeometric mean±standard error at each sampling time for eachexperimental group of monkeys.

FIG. 23 (A-D). Plasma SHIV-89.6P neutralization titers determined fromplasma samples obtained from the monkeys following SHIV-89.6P challenge.Neutralization was determined with an MT-2 dye exclusion assay.

FIG. 24 (A-C). 89.6P Env-specific PBMC IFN-γ ELISPOT responses assessed1 week following rADV boost and both 3 and 10 weeks following SHIV-89.6Pchallenge. ELISPOT responses were determined after in vitro exposure ofPBMCs (peripheral blood lymphocytes, PBL) to peptide pools spanning theHIV-1 89.6P Env protein. The bars represent the mean values for sixmonkeys with the standard error shown.

FIGS. 25 A, B and C. Vaccine-elicited cellular immune responses to HIV-1clade A, clade B, clade C, and 89.6P Env antigens by PBL of rhesusmonkeys following DNA prime and rAd boost immunizations. PBL werefreshly isolated at weeks 12 (post-DNA prime) (A), 27 (post-rAd boost)(B) and 42 (day of challenge) (C) post-immunization and assessed forIFN-γ ELISPOT responses following stimulation with peptide poolsspanning the indicated HIV-1 Env proteins. Data are presented as themean number of antigen-specific spot forming cells (SFC) per 10⁶PBL+/−SEM from 6 monkeys per group.

FIG. 26. Vaccine-elicited cellular immune responses to SIV Gag and Polby PBL of rhesus monkeys following DNA prime/rAd boost immunizations.PBL were freshly isolated at week 27 post-immunization (1 week followingrAd boost) and assessed for IFN-γ ELISPOT responses followingstimulation with peptide pools spanning the SIV Gag and Pol proteins.Data are presented as the mean number of antigen-specific spot formingcells (SFC) per 10⁶ PBL+/−SEM from 6 monkeys per group.

FIG. 27. Antibody titers to HIV-1 clade A, clade B, or clade C Envproteins in plasma from rhesus monkeys following DNA prime/rAd boostimmunizations. Plasma samples were obtained at week 28 post-immunization(2 weeks following rAd boost) and anti-gp145 antibody titers to theindicated HIV-1 Env proteins were determined by ELISA. Data arepresented as the mean geometric titer from 6 monkeys per group.

FIGS. 28 A, B and C. Antibody neutralizing activity in plasma of rhesusmonkeys following DNA prime/rAd boost immunizations. Plasma samples wereobtained from vaccinated and control monkeys at week 28post-immunization (2 weeks following rAd boost), and tested forneutralizing activity against panels of clade A, clade B, and clade CHIV-1 isolates. The dashed line represents a reference point of 20%neutralization, as noted in the results section. Data are presented asthe mean percent neutralizing activity+/−SEM from 6 monkeys per group.Note that the top panel of clade A viruses also includes a control MuLVEnv pseudovirus.

FIGS. 29 A and B. Cellular immune responses to HIV-1 Env and SIV Gag andPol by PBL of vaccinated and control rhesus monkeys following SHIV-89.6Pchallenge. PBL were freshly isolated two weeks following challenge andassessed for IFN-γ ELISPOT responses following stimulation with peptidepools spanning the indicated HIV-1 Env proteins (A) or the SIV Gag andPol (B) proteins. Data are presented as the mean number ofantigen-specific spot forming cells (SFC) per 10⁶ PBL+/−SEM from 6monkeys per group.

FIGS. 30 A and B. Plasma viral RNA levels following SHIV-89.6Pchallenge. The peak plasma viral RNA level (A) for each monkey wasmeasured on day 16 post-challenge. The set point plasma viral RNA level(B) for each monkey was calculated as the median of values detectedbetween days 85 and 169 post-challenge. Log viral copies/ml fromindividual monkeys are indicated, with bars indicating the median valueof the 6 monkeys per experimental group. The detection limit of theassay, 125 copies/ml, is shown with a dashed line.

FIG. 31. Peripheral blood CD4⁺ T lymphocytes post-SHIV-89.6P challenge.The percentage of CD3⁺CD4⁺ T lymphocytes in the peripheral blood of therhesus monkeys was assessed by flow cytometry through day 169 followingSHIV-89.6P infection. Data are presented as the mean percent ofperipheral blood CD4⁺ T lymphocytes from 6 monkeys per group+/−SEM.

FIG. 32. VRC-4306 DNA construct. This plasmid DNA is designed to expressthe HIV-1 Gag, Pol, and Nef polyproteins with modifications to reducepotential toxicity (deletions in the regions which affect protease, RTand integrase) and increase expression in human cells, together with astrong, constitutive CMV promoter. It contains the gene for kanamycinresistance incorporated into the bacterial vector backbone as aselectable marker.

FIG. 33. VRC-5305 DNA Construct. This plasmid DNA is designed to expressthe HIV-1 Clade A Env protein with modifications to reduce potentialtoxicity (deletions of fusion and cleavage domains and the interspacebetween heptad (H) 1 and 2) and increase expression in human cells,together with a strong, constitutive CMV promoter. It contains the genefor kanamycin resistance incorporated into the bacterial vector backboneas a selectable marker.

FIG. 34. VRC-2805 DNA Construct. This plasmid DNA is designed to expressthe HIV-1 clade B Env glycoprotein with modifications to reducepotential toxicity (deletions of fusion and cleavage domains and theinterspace between heptad (H) 1 and 2) and increase expression in humancells, together with a strong, constitutive CMV promoter. It containsthe gene for kanamycin resistance incorporated into the bacterial vectorbackbone as a selectable marker.

FIG. 35. VRC-5309 DNA Construct. This plasmid DNA is designed to expressthe HIV-1 Clade C Env glycoprotein with modifications to reducepotential toxicity (deletions of fusion and cleavage domains and theinterspace between heptad (H) 1 and 2) and increase expression in humancells, together with a strong, constitutive CMV promoter. It containsthe gene for kanamycin resistance incorporated into the bacterial vectorbackbone as a selectable marker.

FIG. 36. Plasmid map for HIV-1 Clade B Gag (VRC-4401).

FIG. 37. Plasmid map for HIV-1 Clade B Pol (VRC-4409).

FIG. 38. Plasmid map for HIV-1 Clade B Nef (VRC-4404).

FIG. 39. Plasmid map for HIV-1 Clade A Env (VRC-5736).

FIG. 40. Plasmid map for HIV-1 Clade B Env (VRC-5737).

FIG. 41. Plasmid map for HIV-1 Clade C Env (VRC-5738).

FIG. 42. Adgp 140(A).11D adenoviral vector map.

FIG. 43. Adgp 140(C).11D adenoviral vector map.

FIG. 44. B287-B Adt.gp140dv12(B).11D adenoviral vector map.

FIG. 45. GV326A Adt.GagPol(B).11D adenoviral vector map.

FIG. 46. Plasmid map for VRC 5747.

FIG. 47. Plasmid map for VRC 5753.

FIG. 48. Plasmid map for VRC 5754.

FIG. 49. Plasmid map for VRC 5755.

FIG. 50. Plasmid map for VRC 5766.

FIG. 51. Plasmid map for VRC 5767.

FIG. 52. Plasmid map for VRC 5768.

FIG. 53. Plasmid map for VRC 5769.

FIG. 54. Plasmid map for VRC 5770.

FIG. 55. Plasmid map for VRC 5771.

FIG. 56. Plasmid map for VRC 5772.

FIG. 57. Plasmid map for VRC 5773.

FIG. 58. Plasmid map for CMVR-gp145ΔCFIΔV1(V2ΔLR)(V3-1AB)(Bal).

FIG. 59. Plasmid map for CMVR-gp145ΔCFI(V1V2AG)(V3-1AB)(Bal).

FIG. 60. Plasmid map for CMVR-gp145ΔCFI(V1ΔG)(V2ΔLR)(V3-1AB)(Bal).

FIG. 61. Plasmid map for CMVR-gp145ΔCFI(V1ΔG)(V2ΔM)(V3-1AB)(Bal).

FIG. 62. Plasmid map for CMVR-145ΔCFI(V1ΔG)ΔV2(V3-1AB)(Bal).

FIG. 63. Plasmid map for CMVR-gp145ΔCFI(V1ΔLR)(V2AG)(V3-1AB)(Bal).

FIG. 64. Plasmid map for CMVR-gp145ΔCFI(V1ΔLR)ΔV2(V3-1AB)(Bal).

FIG. 65. Plasmid map for CMVR-gp145ΔCFI(V1ΔM)(V2AG)(V3-1AB)(Bal).

FIG. 66. Plasmid map for CMVR-gp145ΔCFI(V1ΔM)ΔV2(V3-1AB)(Bal).

FIG. 67. Plasmid map for CMVR-gp145ΔCFI(V3-1AB)(Bal).

FIG. 68. Plasmid map for CMVR-gp145ΔCFIΔV1(V2ΔG)(V3-1AB)(Bal).

FIG. 69. Plasmid map for CMVR-gp145ΔCFIΔV1(V2ΔM)(V3-1AB)(Bal).

FIG. 70. Plasmid map for CMVR-gp145ΔCFIΔV1(V3-1AB)(Bal).

FIG. 71. Plasmid map for CMVR-gp145ΔCFIΔV1V2(V3-1AB)(Bal).

FIG. 72. Plasmid map for CMVR-gp145ΔCFIΔV2(V3-1AB)(Bal).

FIG. 73. Adenoviral vector map for VRC 5781.

FIG. 74. Plasmid map for VRC 5782.

FIG. 75. Adenoviral vector map for VRC 5783.

FIG. 76. Plasmid map for VRC 5784.

FIG. 77. Adenoviral vector map for VRC 5785.

FIG. 78. Plasmid map for VRC 5786.

FIG. 79. Adenoviral vector map for VRC 5787.

FIG. 80. Plasmid map for CMVR-gp145ΔCFI(BBBB).

FIG. 81. Adenoviral vector map for VRC 5789.

FIG. 82. Plasmid map for VRC 5790.

FIG. 83. Adenoviral vector map for VRC 5791.

FIG. 84. Plasmid map for VRC 5792.

FIG. 85. Adenoviral vector map for VRC 5793.

FIG. 86. Plasmid map for VRC 5794.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Part I Expanded Breadthof Virus Neutralization after Immunization with a Multiclade EnvelopeHIV Vaccine Candidate

Abstract

Although the V3 loop of the human immunodeficiency virus type 1 (HIV-1)envelope (Env) effectively elicits potent neutralizing antibodyresponses, the specificity of the antibody response is often restrictedto T cell line adapted (TCLA) strains and a small subset of primaryisolates, limiting its utility for an AIDS vaccine. In this study, wehave compared Env immunogens with substituted V3 regions to combinationsof strains from different clades and evaluated their ability to expandthe breadth of the neutralizing antibody response. When the V3 regionfrom HIV BaL was substituted for HIV HXB2, an effective neutralizingantibody response against several clade B primary isolates was elicited,but it remained restricted to neutralization of mostly clade B isolates.In an attempt to expand this response further, a linear epitoperecognized by the broadly neutralizing 2F5 antibody was inserted intoV3. A V3 2F5 epitope was identified that bound to 2F5 and elicited apotent 2F5 antibody response as an immunogen, but the antiseraneutralized only a lab-adapted strain and not primary isolates. Incontrast, combinations of Envs from clades A, B, and C, elicitedneutralizing antibodies to a more diverse group of primary HIV-1isolates. These studies indicate that combinations of Env immunogens,despite the limited reactivity of the V3 from each component, can beused to expand the breadth of the neutralizing antibody response.

Introduction

Significant advances have been made in the development of AIDS vaccinecandidates that elicit cell-mediated immune responses, and theseresponses contribute to natural and vaccine-induced immune protectionagainst disease (reviewed in Letvin, N. L. & Walker, B. D. 2003 Nat Med9:861-6). At the same time, it is reasonable to expect that broadlycross-reactive and potent neutralizing antibody responses could play amajor role in protective immunity to HIV. For example, several humanmonoclonal antibodies have been identified that can neutralize a broadspectrum of primary isolates (Burton, D. R. et al. 1994 Science266:1024-7; Conley, A. J. et al. 1994 PNAS USA 91:3348-52; Gauduin, M.C. et al. 1997 Nat Med 3:1389-93; Kessler, J. A. et al. 1997 AIDS ResHum Retroviruses 13:575-82; Muster, T. et al. 1994 J Virol 68:4031-4;Trkola, A. et al. 1995 J Virol 69:6609-17). These antibodies can beprotective if administered at high concentration shortly before viralchallenge (Baba, T. W. et al. 2000 Nat Med 6:200-6; Conley, A. J. et al.1996 J Virol 70:6751-8; Mascola, J. R. et al. 1999 J Viral 73:4009-18;Mascola, J. R. et al. 2000 Nat Med 6:207-10; Parren, P. W. et al. 1995AIDS 9:F1-F6; Parren, P. W. et al. 2001 J Virol 75:8340-7; Shibata, R.et al. 1999 Nat Med 5:204-10). A variety of factors may determinewhether antibodies elicited by envelope (Env) immunogens react with thenative trimeric Env glycoproteins sufficiently to neutralize virus.Attempts have been made to modify this glycoprotein to retain itsoligomeric native structure in an effort to elicit such antibodies(Barnett, S. W. et al. 2001 J Viral 75:5526-40; Chakrabarti, B. K. etal. 2002 J Viral 76:5357-68; Earl, P. L. et al. 2001 J Viral 75:645-53;Lee, S. A. et al. 201 Vaccine 20:563-76; Lund, O, S. et al. 1998 AIDSRes Hum Retroviruses 14:1445-50; Schonning, K. et al. 1998 AIDS Res HumRetroviruses 14:1451-6; Schulke, N. et al. 2002 J Virol 76:7760-76;Srivastava, I. K. et al. 2002 J Virol 76:2835-47; Srivastava, I. K. etal. 2003 J Virol 77:2310-20).

Though it appears that highly conserved epitopes in different HIV-1strains are accessible to antibodies, it is difficult to elicit antibodyresponses to them. Among the established broadly neutralizing monoclonalantibodies, the 2F5 epitope is linear in nature and is found in theectodomain of gp41 (Muster, T. et al. 1993 J Virol 67:6642-7; Purtscher,M. et al. 1994 AIDS Res Hum Retroviruses 10:1651-8; Stiegler, G. et al.2001 AIDS Res Hum Retroviruses 17:1757-65; Zwick, M. B. et al. 2001 JVirol 75:10892-905). Antibodies to this region have been found rarely inHIV-1 seropositive individuals, indicating that this epitope is poorlyimmunogenic. Attempts have been made previously to insert the 2F5epitope into the V3 loop of gp120 to increase Env immunogenicity,without success (Liang, X. et al. 1999 Vaccine 17:2862-72). We havereported modifications of the envelope glycoprotein that increase theantibody response to Env (Chakrabarti, B. K. et al. 2002 J Viral76:5357-68). A modified form of HIV-1 Env with mutations in the cleavagesite, fusion peptide and interhelical regions (ΔCFI), has been shown toimprove the antibody response while maintaining its ability to inducevirus-specific cytotoxic T lymphocytes. In these vectors and a number ofprotein immunogens, the V3 region is particularly immunogenic andelicits potent, although restricted, antibody responses.

In this study, we have examined the ability of the V3 loop to elicitbroadly neutralizing antibody responses. Two approaches have beentaken: 1) introduction of heterologous sequences into the V3 loop, and2) inclusion of multiple envelopes from different clades in the vaccine.As a model for insertion of heterologous sequences, the 2F5 epitope wasanalyzed. For the inclusion of multiple V3 Envs, a combination of cladesA, B, and C was evaluated. Though the positionally inserted 2F5 epitopein ΔCFI Env elicited antibody against the linear peptide, it did notneutralize primary virus isolates. In contrast, the multiple clade Envimmunogen helped to expand the immune response to several strains testedfrom these alternative clades. The combination of HIV envelope genesfrom different clades induced neutralizing antibody to a number ofunrelated lab-adapted strains and primary isolates. These studiessuggest that the V3 loop can contribute to Env antibody immunogenicity,and combination Env immunization can expand the breadth of theneutralizing antibody response in an HIV vaccine candidate.

Materials and Methods

Immunogens. Plasmids encoding CCR5-tropic V3 loops from clades A, B andC were built on the backbone of gp145ΔCFI and gp140ΔCFI versions of theCXCR4-tropic strain HIV HXB2 (GenBank accession number K03455) and theCCR5-tropic strain HIV BaL (GenBank accession number K03455) asdescribed previously (Chakrabarti, B. K. et al. 2002 J Virol76:5357-68). Briefly, to produce a CCR5-tropic version of the envelopeglycoprotein (CCR5 gp160/h), the region encoding amino acids 205 to 361from HIV HXB2 gp160 was replaced with the corresponding region from theHIV BaL strain of HIV-1 (GenBank accession number M68893, with preferredhuman codon usage) to make it hybrid, HIVHXB/Bal. Synthetic versions ofclades A and C gp145ΔCFI and gp140ΔCFI Env glycoprotein were made basedon HIV-1 strains 92rw020 (CCR5-tropic, GenBank accession number U51283)and 97ZA012 (GenBank accession number AF286227) following the sameapproach described above. The fusion domain and the cleavage sequencefrom amino acids 486-519 and the interspace between H1(heptad 1) and H2(heptad 2) from amino acids 576-604 were deleted and the protein wasterminated after the codons for aa 690 and aa 664 to make gp145ΔCFI andgp140ΔCFI Env respectively. The fusion domain and the cleavage sequencefrom amino acids 487-520 and the interspace between H1 and H2 from aminoacids 577-605 Glade C gp160 were deleted. The protein was terminatedafter the codons for aa 689 and aa 664 to create a synthetic proteinclade C gp145ΔCFI/h and clade C gp140ΔCFI/h respectively. For the 2F5 V3chimeric Envs, a hybrid envelope gp140ΔCFI B(C-HR2), completely lacking2F5 (−2F5), was made by replacing the sequence of CCR5-tropic gp140ΔCFIof strain of HIVHXB/Bal from aa 592 to 680 that includes the HR2 (heptadrepeat 2) and the monoclonal antibody 2F5 binding region with thecorresponding region from clade C gp140ΔCFI that lacks 2F5, aa 592 to688. The hybrid envelope gp140ΔCFI clade B (C-HR2), -2F5, in HIVHXB/Balbackbone was further modified by deleting GPGRA (aa 309-313) to generategp140ΔCFIAGPGRA B(C-HR2), designated -tip -2F5. The minimal and theextended 2F5 epitopes encoding ‘LELDKWAS’ (SEQ ID NO: 3) and‘KNEQELLELDKWAS’ (SEQ ID NO: 4) respectively were inserted in the placeof GPGRA (SEQ ID NO: 12) in the V3 loop of gp140ΔCFI B(C-HR2), -2F5, bysite-directed mutagenesis to form gp140ΔCFI B(C-HR2) 2F5, termed V3 2F5,and gp140ΔCFI B(C-HR2) ext 2F5 or V3 ext2F5 respectively.

Expression analysis of envelope proteins in transfected cells.Expression of Envs was confirmed as described previously by transfectionand Western blotting in 293 cells (Chakrabarti B K et al. 2002 J Virol76:5357-68). Binding to soluble CD4 (sCD4) was performed as describedpreviously (Chakrabarti, B. K. et al. 2002 J Virol 76:5357-68; Karlsson,G. B. et al. 1998 J Exp Med 188:1159-71). The abilities of severalmonoclonal antibodies, 2F5, 2G12, F105, and IgG1b12, to bind gp140ΔCFIfrom different clades were determined as described previously(Chakrabarti, B. K. et al. 2002 J Viral 76:5357-68). Antibody (5 μg) wasused to immunoprecipitate gp140ΔCFI from 100 μl of membrane-freesupernatant from 293 cells transfected with the expression vectorexpressing clade A, clade B or clade C gp140ΔCFI. The same volume ofsupernatant from cells transfected with empty vector was used as acontrol. Antibodies were obtained from the AIDS Research and ReferenceReagent Program, National Institutes of Health. The binding of HIV-1 IgGto either R5/B 140ΔCFI or different mutants was measured by ELISA.Briefly, Immulon 2HB ELISA plates (Thermo Labsystems, Franklin, Mass.)were coated with 100 μl/well of Lectin Galanthus Nivalis (Sigma, St.Louis, Mo.) (10 μg/ml in PBS) overnight at 4° C. The plates were blockedwith 200 μl of PBS containing 10% FBS for 2 hours at room temperature,and washed twice with PBS containing 0.2% TWEENT™-20 (PBS-T). Sampleswere added and developed as described in PART II below. To detect the2F5 or V3 antibodies in sera of immunized guinea pigs, ELISA plates werecoated with either 100 μl of 2F5 peptide, KNEQELLELDKWAS (10 μg/ml) (SEQID NO: 4), or 100 μl of V3 peptide, ‘TRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAH’(SEQ ID NO: 13), overnight at 4° C. The peptide solution was removedfrom the wells and blocked with 200 μl of PBS containing 10% FBS for 2hours at room temperature. The plates were washed twice with PBScontaining 0.2% TWEEN™ 20 (PBS-T), and then the sera from immunizedguinea pigs from different groups were added with 3-fold dilutions for 1hour.

Immunizations. Six-week-old female Huntley guinea pigs were injectedintramuscularly with 500 μg of purified plasmid DNA encoding thegp145ΔCFI forms of the relevant immunogens in 400 μl of normal saline.For multiclade A, B, and C envelope immunization, one-third of total 500μg of DNA was used for each envelope expressing plasmid. For eachplasmid DNA, a group of four guinea pigs was injected three times atintervals of 2 weeks. The guinea pigs were bled 2 weeks after the lastinjection, and sera were collected and stored at 4° C. The guinea pigsreceived a boost with replication-defective recombinant adenovirus (ADV)encoding the gp140ΔCFI form of the same immunogen as describedpreviously (Sullivan, N. J. et al. 2000 Nature 408:605-9; Xu, L. et al.1998 Nat Med 4:37-42; Yang, Z. et al. 1998 Science 279:1034-7) and werebled 2 weeks after ADV injection.

HIV-1 Viruses. HIV-1 primary isolates, and the T-cell line adapted HIVMN and HIV IIIB, were obtained from NIH AIDS Research and ReferenceReagent Program except as noted below. Primary isolates 6101 (previouslycalled P15) and 1168 are CCR5 using clade B HIV-1 strains describedpreviously (Bures, R. et al. 2000 AIDS Res Hum Retroviruses 16:2019-35).DU151, DU123 and 5007 are Glade C viruses that have also been previouslydescribed (Bures R et al. 2002 J Virol 76:2233-44). TV1 (Glade C) wasprovided by Estrelita Janse Van Rensburg (University of Stellenbosch,South Africa). DJ263 is a clade A virus that was provided byinvestigators from the U.S. Military HIV Research Program. All primaryviral stocks were prepared and titrated in PHA and IL-2 stimulated humanperipheral blood mononuclear cells (PBMC). Viruses BL01 and BR07 wereprovided by Dana Gabuzda of the Dana-Farber Cancer Institute (Ohagen, A.et al. 2003 J Virol 77:12336-45). Both are chimeric infectious molecularclones of NL4-3 that contain the near full-length env genes from HIV-1strains indicated. After initial plasmid transfection of 293 cells,these viruses were expanded in PBMC as described above.

Neutralizing antibody assays. Two assays for neutralization were used.Neutralization of a BaL isolate was measured in PBMC by using areduction in p24 Gag antigen synthesis as described previously (Bures,R. et al. 2000 AIDS Res Hum Retroviruses 16:2019-35). Briefly, 500 50%tissue culture infective doses of virus were incubated with variousdilutions of test samples (serum) in triplicate for 1 h at 37° C. in96-well U-bottom culture plates. PHA-PBMC were added and incubated forone day. The cells were then washed three times with growth medium andresuspended in 200 μl of fresh growth medium. Culture supernatants (25μl) were collected twice daily thereafter and mixed with 225 μl of 0.5%Triton X-100. The 25 μl of culture fluid removed each day was replacedwith an equal volume of fresh growth medium. Concentrations of p24 Gagantigen were measured in an antigen capture ELISA as described by thesupplier (DuPont/NEN Life Sciences, Boston, Mass.). Concentrations ofp24 in virus control wells (virus plus cells but no test serum) weredetermined for each harvest day. Concentrations in all remaining wellswere determined for a harvest day that corresponded to a time when p24production in virus control wells was in an early linear phase ofincrease that exceeded 3 ng/ml, which is when optimum sensitivity isachieved in this assay (Zhou, J. Y. & Montefiori, D. C. 1997 J Virol71:2512-7). The limit of detection in the p24 ELISA was 0.1 ng ofp24/ml. Neutralization titers are given as the reciprocal of the minimumserum dilution (calculated prior to the addition of cells) that reducedp24 synthesis by 80% relative to a negative control serum sample from ahealthy, HIV-1-negative individual. Neutralization assay for TCLAstrains were performed in either MT-2 cells (HIV IIIB and HIV MN) byusing neutral red to quantify the percentage of cells that survivedvirus-induced killing (Montefiori, D. C. et al. 1988 J Clin Microbiol26:231-5). Briefly, 500 50% tissue culture infective doses of virus wereincubated with multiple dilutions of serum samples in triplicate for 1 hat 37° C. in 96-well flat-bottom culture plates. Cells were added andthe incubation continued until most but not all of the cells in viruscontrol wells (cells plus virus but no serum sample) were involved insyncytium formation (usually 4 to 6 days). Cell viability was quantifiedby neutral red uptake as described (Montefiori, D. C. et al. 1988 J ClinMicrobiol 26:231-5). Neutralization titers are defined as the reciprocalserum dilution (before the addition of cells) at which 50% of cells wereprotected from virus-induced killing. A 50% reduction in cell killingcorresponds to an approximate 90% reduction in p24 Gag antigen synthesisin this assay (Bures, R. et al. 2000 AIDS Res Hum Retroviruses16:2019-35). Each set of assays included a positive control serum thathad been assayed multiple times and had a known average titer.V3-specific neutralizing antibodies were assessed by incubating dilutedserum samples (diluted with an equal volume of phosphate-bufferedsaline, pH 7.4) for 1 h at 37° C. in the presence and absence of V3peptide (50 μg/ml). Titers of neutralizing antibodies were thendetermined in either the PBMC assay (in the case of primary isolates) orthe MT-2 cell assay (in the case of HIV MB and HIV MN) by using neutralred as described above.

The alternative assay used a single round intracellular p24-antigen flowcytometric HIV-1 neutralization assay has been described previously(Mascola, J. R. et al. 2002 J Virol 76:4810-21). Briefly, 40 μl of virusstock (multiplicity of infection, approximately 0.1) was incubated with10 μl of heated inactivated guinea pig serum, or with 10 μl of controlantibody. After incubation for 30 min at 37° C., 20 μl of mitogenstimulated CD8 depleted PBMC (1.5×10⁵ cells) were added to each well.These T-cells were maintained in IL-2 culture medium containing 1 μMindinavir, and the cells were fed on day 1 with 150 μl of IL-2 culturemedium. One day after infection, cells were stained for intracellularp24-Ag using the KC57 (Beckman Coulter, Inc.) anti-p24 antibody,followed by quantitation of HIV-1 infected cells by flow cytometry. Livecells initially gated by forward and side scatter were analyzed forp24-Ag positive cells. After forward and side scatter gating, 50,000events were counted. Final quantitation of p24 positive PBMC was done bysubtraction of background events in mock-infected cells (typically lessthan 10 cells per 50,000 events counted). The percent neutralization wasderived by calculating the reduction in the number of p24-Ag positivecells in the test wells with immune sera, compared to the number ofp24-Ag positive cells in wells containing pre-immune sera from thecorresponding animal. All assays included additional control wells withcommercial pooled guinea pig sera (Gemini Bio-Products, Woodland,Calif.), as well as positive control wells containing well characterizedmonoclonal or polyclonal neutralizing antibodies. Standard operatingprocedures prescribed the acceptable positive and negative controlvalues, and all data shown are from assays that met these criteria.

Peptide competition assays were done in the same assay format, exceptthat the V3 peptide was added to the serum 30 minutes before virus wasadded. The concentration of peptide reported was that present whenpeptide, serum and virus were incubated together. The V3 peptides basedon HIV-1 strains BaL, ZA12 and RW20 (matching the vaccine strains), anda scrambled V3 peptide, was made as a 23mer (IGPGRATRPNNNFYTTGTRKSIH)(SEQ ID NO: 14) by SynPep (Dublin, Calif.). The HIV IIIB V3 peptide (a24 mer) was purchased from Sigma-Aldrich. The scrambled V3 peptide wasincluded as a control in all assays. Additional controls, a mixture of22 peptides (15 mers overlapping by nine spanning the Ebola (Zaire)viral glycoprotein sequence), were used to confirm specificity of V3peptide inhibition.

Results

Generation of V3 loop Modified Env Immunogens. To develop HIV Envvaccines with alternative V3 specificities, modifications of a previousclade B HIV-1 prototype strain (Chakrabarti, B. K. et al. 2002 J Virol76:5357-68) containing ΔCFI mutations were made. Replacements of the V3loop were made at the junction of highly conserved sites at the base ofC2 and C4. Specifically, the V3 loop of HIV HXB2 was replaced with thatof a CCR5-tropic strain, HIV BaL (FIG. 1A). Expression of these envelopeglycoproteins was confirmed in transfected 293 cells as visualized byWestern blot analysis (FIG. 1B). As with earlier prototypes(Chakrabarti, B. K. et al. 2002 J Virol 76:5357-68), the gp140ΔCFI,which lacks the transmembrane domain, was readily detected in thesupernatant, indicating that it could give rise to soluble antigen.

Induction of neutralizing antibody responses by Env immunogen, HIVHXB/Bal. Sera from guinea pigs immunized with the HIV HXB/BaL gp140ΔCFIimmunogen were able to neutralize laboratory-adapted strains HIV MN, toa lesser extent, HIV IIIB (FIG. 2A) and CCR5-tropic HIV-1 BaL (FIG. 2B).In contrast, sera from guinea pigs immunized with the parental HIV HXBgp140ΔCFI were not able to neutralize these viruses. It was thereforepossible to generate neutralizing antibodies against HIV BaL byinserting the V3 loop of this virus in place of the HIV HXB2 V3 loopthat existed in the gp140ΔCFI immunogen. To determine whether theneutralizing activity was mediated by anti-V3 antibodies, competitionassays were performed using peptides corresponding to the V3 loop ofeither HIV BaL or HIV MB. This analysis revealed that theantibody-mediated neutralization of HIV BaL was largely V3-dependent(FIG. 3A), as it was inhibited by the HIV BaL but not by the HIV IIIB V3peptide (FIG. 3A). Neutralization of HIV MN by sera from guinea pigsimmunized with parental HIV HXB/BaL gp140ΔCFI was also shown to be V3dependent (FIG. 3B).

Insertion of the 2F5 epitope into the V3 region. Because the breadth ofneutralization by these V3 substitutions remained limited, we askedwhether it was possible to insert an epitope into the V3 region that wasrecognized by a broadly neutralizing antibody. The linear epitope forthe antibody, 2F5, represents such a well-defined peptide sequence. Amodification was made in the ectodomain of gp41, replacing the clade BHR2 (heptad repeat 2) with the homologous clade C HR2 (heptad repeat 2),which lacks the sequence that is recognized by 2F5 monoclonal antibody,termed -2F5 (FIG. 4A). In this way, the effect of the epitope for 2F5 inthe V3 region alone could be assessed. A mutant V3 loop sequence wasprepared in which the sequence for 2F5 epitope replaced native V3sequence at the tip of the V3 loop, designated V3 2F5. The tip of V3,GPGRA (SEQ ID NO: 8), was deleted in another version, -tip -2F5, as anegative control (FIG. 4A). The minimal peptide that is recognized by2F5 antibody, defined previously (Muster T et al. 1994 J Virol68:4031-4), as well as an extended amino acid sequence more recentlyrecognized (Zwick M B et al. 2001 J Virol 75:10892-905), were insertedin the B (C-HR2), V3 ext2F5. Expression of the 2F5 epitope inserted inV3 was confirmed in transfected 293 cells by Western blot analysis. Theexpression level of these V3 derivatives varied, depending on thepresence of the GPGRA tip sequence (FIG. 4B). The expressed proteinbearing the 2F5 epitope in mutant V3 reacted with monoclonal antibody2F5 by ELISA and the gp140ΔCFI B (C-HR2) with the extended 2F5 epitopesequence, V3 ext 2F5, showed 20- to 30-fold higher reactivity than theparental clade B gp140ΔCFI (FIG. 4C, left panel). The various mutant V3′s with 2F5 epitope sequences in gp140ΔCFI envelopes reacted similarlywith HIV-1 IgG (FIG. 4C, right panel). It was therefore possible toincrease the antigenicity of the 2F5 epitope by insertion of thesesequences in the correct position of the V3 loop.

Immunogenicity of the 2F5 V3 loop mutants. Guinea pigs were immunizedwith plasmid DNA and boosted with adenoviral vectors encoding these 2F5epitopes inserted into V3. The wild-type gp140/145 ΔCFI expressionvectors did not elicit antibodies that bound to the peptide containingthe 2F5 epitope, similar to the B (C-HR2), -2F5, negative control thatlacked the sequence altogether (FIG. 5A, B). Similarly, the vectors thatencoded the amino acid sequence for the minimal 2F5 epitope (V3 2F5),despite their ability to bind 2F5 antibodies, did not elicit ameasurable 2F5 like antibody response. In contrast, the gp140□CFI B(C-HR2) with the extended sequence for 2F5 region (V3 ext 2F5) inducedthe production of antibodies in guinea pigs that could recognize thepeptide containing the sequence for extended 2F5 epitope (FIG. 5A, B).These results indicate that insertion of the appropriate sequence for2F5 epitope in V3 renders this epitope immunogenic.

To determine whether these antisera could inhibit diverse HIV isolates,neutralization assays were performed. These antibodies showedsubstantial inhibition of a CXCR4-tropic HIV IIIB isolate. However, theyfailed to inhibit replication of the CCR5-tropic HIV BaL or HIV SF162isolates (FIG. 5C), indicating that these antibodies were not broadlyneutralizing.

Expression and characterization of multiclade Env immunogens. Plasmidexpression vectors encoding clade A and clade C gp140/145ΔCFI proteinswere synthesized using the same modified codon preferences and mutationsapplied to the clade B vectors. Their expression was confirmed intransfected 293 cells by immunoprecipitation with well-defined broadlyneutralizing monoclonal antibodies such as 2F5, 2G12, F105, and IgG1b12,followed by Western blot analysis (FIG. 6A). Reactivity of theseantibodies with clades A, B, and C varied in terms of recognition andspecificity (FIG. 6A) as expected from previous analyses with theseantibodies across clades (Moore, J. P. et al. 1994 J Virol 68:8350-64;Trkola, A. et al. 1996 J Virol 70:1100-8; Kostrikis, L. G. et al. 1996 JVirol 70:445-58). Env derived from clades A and B reacted with 2F5antibody, in contrast to clade C, which showed no detectable reactivityby immunoprecipitation and Western blotting. In contrast, clades A and CEnv readily interacted with IgG1b12, whereas a clade B Env showed weakerreactivity with the same monoclonal antibody. All Envs showed similarbinding to the monoclonal antibody, 2G12. The gp140ΔCFI forms that lackthe transmembrane domain were readily detected in the supernatant (FIG.6A, B), indicating that they gave rise to soluble antigen. To furtherassess whether these glycoproteins retained conformational structuresrelevant to Env function, their ability to interact specifically withits receptor, CD4, was assessed. Compared to negative controlsupernatants, these Envs readily bound to soluble CD4 produced fromtransfected 293 cells (FIG. 6B), as previously described for clade B(Chakrabarti, B. K. et al. 2002 J Virol 76:5357-68), confirming that theCD4 binding site determinants were intact.

Immunization with the multiclade Env vaccine candidate increases thebreadth of the neutralizing antibody response. The ability of themulticlade Env vaccine candidate to elicit neutralizing antibodies wasanalyzed by immunization with an equal mixture of these vectors andcompared to antibodies elicited by the single clade B Env immunogen(monoclade) vaccination as described in Materials and Methods. ELISAswere done using clade-specific envelope captured on lectin-coatedplates, or by using V3 peptides. Our data showed that the antibodyresponse after HxB2/BaL immunization was directed preferentially toclade B Env and clade B V3. In contrast, sera from the multicladeimmunized animals were reactive with clade A, B, and C Env proteins andV3 peptides. Antisera were tested against a panel of 19 viruses (3 cladeA, 11 clade B and 5 clade C). Sera from four guinea pigs immunized withclade B gp140/145ΔCFI immunogen were able to neutralize several clade Bprimary isolates (FIG. 7A). This single-round of infection flowcytometric assay enumerates the number of HIV-1 infected cells and isable to detect fairly low levels of virus neutralization. In all assays,the immune sera were compared directly to the pre-immune sera from thecorresponding animal. While a 1:5 serum dilution of guinea pig seraneutralized some clade B viruses, others were not neutralized at all.Additionally, very little neutralization was observed against the 3clade A and 5 clade C viruses. Importantly, sera from guinea pigsimmunized with a mixture of clade A, B, and C □CFI Envs maintained theirneutralization of clade B viruses (FIG. 7B). Thus, the mixture of threeEnv plasmids did not detract from the immunogenicity of the clade B Env.Additionally, these sera displayed some modest level of neutralizationagainst several non-Glade B viruses (FIG. 7B). A non-parametric MannWhitney test comparing the median percent neutralization value of thetwo groups (monoclade vs. multiclade) for each non-clade B virus wasperformed. The p value was less than 0.05 for virus isolates DJ263,ZAl2, TV1, and DU151. Thus, for these viruses, the breadth ofneutralization by the multiclade sera was significantly greater than themonoclade sera. Of note, sera from the clade B immunized guinea pigswere able to neutralize several clade B isolates with a V3 loop sequencethat was divergent from the homologous BaL immunogen. Neutralization ofclade B HIV BR07 and HIV 89.6 was also observed, despite the fact thatthese two viruses vary from HIV BaL by 10 aa and 8 aa respectively inthe V3 region. Furthermore, this neutralization was V3-mediated, as itwas blocked by HIV BaL V3 peptide where it remained unaffected bycontrol peptides derived from Ebola GP (FIG. 7C).

To determine the contribution of anti-V3 antibodies specificity to virusneutralization, competition studies were also performed using HIV SF162.This clade B virus was chosen because it is a fairly sensitive primaryisolate that was neutralized by sera from both the clade B andmulticlade immune sera. The HIV BaL V3 peptide was able to blockessentially all neutralization of the clade B immune sera. Thus, anti-V3antibodies largely mediated neutralization of HIV SF162 (FIG. 7D). Whileclade B-induced neutralization was abolished by HIV BaL V3 peptide,neutralization of the same isolate by sera from guinea pigs immunizedwith multiclade envelopes was much less sensitive to inhibition by theHIV BaL clade B V3 peptide, indicating that this neutralization wasmediated by non-V3 antibodies, or by V3 antibodies that were notcompeted by the clade B BaL V3 peptide. To address this laterpossibility, we did further competition studies using clade A and C V3peptides that matched the vaccine strains compared to no peptide orscrambled V3 peptide as controls. The addition of clade A and Cpeptides, or a combination of the A, B and C peptides together, producedonly a small decrement in the neutralization of HIV SF162 (FIG. 7E).These data, from one guinea pig, are representative of all four guineapigs in each group. Also, the Glade A and C V3 peptides were able toblock neutralization of some V3 sensitive clade A and C viruses. Thisresult further supports the notion that the multiclade immunogen inducesnon-V3 dependent neutralizing antibodies. Regarding neutralization ofnon-clade B viruses, the modest levels of neutralization observed madeV3 competition studies difficult to perform, but this is the subject offurther study.

Discussion

Based on the ability of CTL to control viremia and protect against theprogression of HIV disease (reviewed in Letvin, N. L. & Walker, B. D.2003 Nat Med 9:861-6), an effective AIDS vaccine will need to induce astrong cell-mediated immune response. For such a vaccine to be highlyeffective and to induce sterilizing immunity, it will likely also benecessary to elicit broadly neutralizing antibodies. There isconsiderable diversity of HIV strains throughout the world, 90% of whichfall into those designated as clades A, B, and C. For these reasons,gene-based vaccines encoding representative candidates from each ofthese clades have been analyzed in this study for their ability toinduce a neutralizing antibody response. We have characterizedcell-mediated immune responses and shown that these multiclade vaccinesinduce Env-specific CD4 and CD8 immune responses to multiple clades inmice (see PART II below). Here, we find that this multiclade vaccinepermits the synthesis of native conformations of Env that induceantibodies with broader reactivity than monoclade immunogens.

For a globally effective vaccine, the cellular and humoral immunity mustrespond to multiple strains from these clades. The candidates developedhere build on previous Env modifications that elicited more potentantibody responses while retaining their ability to stimulateEnv-specific CTL (Chakrabarti, B. K. et al. 2002 J Virol 76:5357-68).These ΔCFI mutations were introduced into clades A and C, which retainedtheir reactivity with known neutralizing antibodies and CD4, as well astheir ability to form trimers, thus preserving physiologically relevantepitopes (FIG. 6). Importantly, all three Env constructs wereimmunogenic and we observed no antigenic interference compared to themonoclade immunization. Interestingly, while monoclade vaccinationinduced neutralizing activity that was competed by homologous HIV BAL V3peptides, indicating that the V3 is the main epitope for elicitingneutralizing antibody, immunization with the multiclade Env elicitedantisera that showed broader reactivity and was less V3-dependent. Thus,while neutralization of non Glade B viruses was quite modest, thismulticlade vaccine approach appeared to expand the breadth ofneutralizing response. An alternative strategy, using a linear epitopeof 15 amino acids that is the target of the broadly neutralizing 2F5antibody, was less successful. This peptide sequence was inserted intothe V3 region of ΔCFI Env to increase its immunogenicity. Though bindingantibodies were elicited, these antibodies failed to neutralize virus,indicating that other interactions of 2F5 contribute to virusneutralization.

As the HIV-1 pandemic continues to grow, increasing numbers ofrecombinant strains have been reported (Kuiken, C. et al. 2000 HumanRetroviruses and AIDS 1999. Los Alamos, N. Mex.: Los Alamos NationalLaboratory) and such viruses continually mutate and escape host immuneresponses (Barouch, D. H. et al. 2002 Nature 415:335-9; Mortara, L. etal. 1998 J Virol 72:1403-10) throughout infection. There has beenconsiderable discussion about the choice of strains to use for candidatevaccines based on genetic relatedness to incident strains (Korber, B. etal. 2000 Science 288:1789-96; Robertson, D. L. et al. 2000 Science288:55-6; Klausner, R. D. et al. 2003 Science 300:2036-9). While thisselection would seem more important if only a single Env immunogen isutilized in a vaccine, it is less compelling when representatives of themajor clades are included within vaccines. For the vaccine strainsutilized in this study, the amino acids sequence of the clade A Env is86% conserved relative to the ancestral and 87% to the consensus A aminoacids sequences, the amino acids sequence of clade B is 88% homologousto the ancestral and 87% to the consensus B sequences, and the aminoacids sequence of clade C is 88% similar to the ancestral C and 87% toconsensus C (hiv.lanl.gov). These vaccine components are thereforereasonably representative of viruses from the major glade designations.Because they were derived from CCR5-tropic isolates, they were likely toretain functional epitopes relevant to viral infection, as confirmed bybinding to neutralizing antibodies and CD4 (FIG. 6).

A multiclade immune response is envisioned to help to reduce thelikelihood of viral escape, both from CTL and antibodies (Richman, D. D.et al. 2003 PNAS USA 100:4144-9; Wei, X. et al. 2003 Nature 422:307-12).

Part II Immunogenicity of Multiple Gene and Clade HIV-1 DNA Vaccines

Abstract

The ability to elicit an immune response to a spectrum of humanimmunodeficiency virus type 1 (HIV-1) gene products from divergentstrains is a desirable feature of an AIDS vaccine. In this study, wehave examined combinations of plasmids expressing multiple HIV-1 genesfrom different clades for their ability to elicit humoral and cellularimmune responses in mice. Immunization with a modified Env, gp145ΔCFI incombination with a Gag-Pol-Nef fusion protein plasmid elicited similarCD4+ and CD8+ cellular responses to immunization with either vectoralone. Further, when mice were immunized with a mixture of Env fromthree clades, A, B, and C, together with Gag-Pol-Nef, the overallpotency and balance of CD4+− and CD8+− T-cell responses to all viralantigens were similar, with only minor differences noted. In addition,plasmid mixtures elicited antibody responses comparable to those fromindividual inoculations. These findings indicate that a multigene andmulticlade vaccine, including components from A, B, C Env andGag-Pol-Nef, can broaden antiviral immune responses without immuneinterference. Such combinations of immunogens are envisioned to helpaddressing concerns about viral genetic diversity for a prospectiveHIV-1 vaccine.

Introduction

The genetic variation of HIV-1 has created challenges for thedevelopment of a preventive AIDS vaccine (van der Groen, G. et al. 1998AIDS Res Hum Retroviruses 14 Suppl 3:S211-S221). Not only would such avaccine be expected to be safe and immunogenic, it must also induceimmune recognition of a broad spectrum of HIV isolates to prove highlyeffective (Mascola, J. R. & Nabel, G. J 2001 Curr Opin Immunol13:489-495). Though progress has been made with subtype-specific andGag- or Env-based HIV vaccines (Bojak, A. et al. 2002 Vaccine20:1975-1979; Deml, L. et al. 2001 J Virol 75:10991-11001; Srivastava,I. K. et al. 2003 J Virol 77:2310-2320), an alternative approachinvolves the utilization of multiple viral proteins from differentclades that can maximize the breadth and potency of the antiviral immuneresponse. An unresolved question for the development of such amultivalent HIV vaccine is whether this approach can elicit strongimmune responses against individual gene products withoutcross-interference. In previous HIV vaccine studies, some multivalentDNA vaccine approaches induced suboptimal immune responses, likely dueto interference among different viral antigens (Kjerrstrom, A. et al.2001 Virology 284:46-61; Muthumani, K. et al. 2002 Vaccine20:1999-2003). In this study, we have addressed this question by usinggene-based vaccination techniques previously used in a variety ofdifferent vaccine studies (Bonnet, M. C. et al. 2000 Immunol Lett74:11-25; Moss, B. 1996 PNAS USA 93:11341-11348; Nabel, G. J. 2001Nature 410:1002-1007; Ramsay, A. J. et al. 1997 Immunol Cell Biol75:382-388).

Env is a major target of both humoral and cellular immunity, while theviral genes for Gag, Pol and Nef are potential targets of the CD8⁺immune response. A modified form of HIV-1 envelope (Env), gp145ΔCFI, hasbeen shown to improve antibody responses while maintaining its abilityto induce cytotoxic T-lymphocyte (CTL) responses (Chakrabarti, B. K. etal. 2002 J Virol 76:5357-5368). A fusion protein of Gag and Pol has alsobeen developed that generates a protein from a single open reading framethat can be processed to present linear epitopes from at least fourviral gene products: Gag, protease (PR), reverse transcriptase (RT), andintegrase (IN) (Huang, Y. et al. 2001 J Viral 75:4947-4951). To ensurethat the poi region did not function in vivo, three point mutations wereintroduced, in PR, RT and IN, termed Pol(ΔPR ΔRT ΔIN). An additionalviral protein, Nef, was included to expand its breadth, andrepresentatives of Clades A, B and C were also generated.

The present study evaluates the immunogenicity of Env and Gag-Pol-Nefvaccine candidates alone or in combination. In addition, the ability tocombine these immunogens from different clade isolates has also beenevaluated. The combination of Gag-Pol-Nef with Env elicited strong CD8immunity to Env without compromising the CD4 or antibody response. Inaddition, combinations of Env from multiple clades help to expand theimmune response to these alternative clades. The combination of multipleHIV genes from different clades is envisioned to facilitate thegeneration of immune responses to diverse HIV strains.

Materials and Methods

Gag-Pol-Nef Immunogens. Plasmids expressing HIV genes were synthesizedby reverse translation (Genetics Computer Group, Inc., Madison, Wis.) ofpublished sequences using codons expected for human cells. The methodsused to make DNA plasmids expressing HIV-1 Gag-Pal-Nef polyproteins fromdifferent clades were similar to those previously described for Gag-Pol(Huang, Y. et al. 2001 J Virol 75:4947-4951). To further inactivateviral proteins, additional inactivating mutations were inserted intoprotease (PR), reverse transcriptase (RT), and integrase (IN). The aminoacid sequence of the Nef protein was not modified, but the NH₂-terminalmyristylation site required for its functional activity was notavailable, as it is synthesized as a fusion protein. The clade A, B andC Gag-Pol-Nef plasmids were 9783, 9790 and 9786 nucleotides in length,respectively, and the clade A, B, and C Env plasmids are 6836, 6869 and6829 nucleotides.

These genes were synthesized by preparation of oligonucleotides of 75base pairs overlapping by 25, or 60 base pairs overlapping by 20, andassembled by Pwo (Boehringer Mannheim) and Turbo Pfu (Stratagene) asdescribed previously (Chakrabarti, B. K. et al. 2002 J Virol76:5357-5368; Huang, Y. et al. 2001 J Viral 75:4947-4951). The cDNAswere cloned into the expression vector pVR1012 (Chakrabarti, B. K. etal. 2002 J Virol 76:5357-5368; Yang Z et al. 1998 Science279:1034-1037). The protein sequence of each Gag polyprotein from theappropriate HIV-1 clade was used to create a synthetic version of thegag gene (gag/h) using codons preferred for expression in human cells.The synthetic gag/h gene contained all mature Gag proteins except for p1and p6 (amino acids 433-500). The synthetic gag/h gene from clade A, B,or C was ligated in frame with codon-modified pol (pol/h) encoding aminoacids 3-1003 from NL4-3 (GenBank accession number M19921). To inactivatethe fusion proteins further, a protease (PR) mutation (Arg to Gly) wasinserted at aa 553, a reverse transcriptase (RT) mutation (Asp to His)at aa 771, and an integrase (IN) mutation (Asp to Ala) at aa 1209. Asynthetic nef gene (nef/h) based on aa 1 to 206 from NL4-3 was fused tothe 3′ end of pol/h by PCR to generate the appropriate Gag-Pol-Nefexpression vector.

For the clade A Gag-Pol-Nef fusion protein, amino acids 1 to 432 from aCCR5-tropic clade A (GenBank accession number AF004885) were used andfused to the pol/h gene described above. In all three Gag-Pol-Nefplasmids, the same poi sequence was inserted, as this viral gene productis more than 90% conserved at the amino acid level among disparateclades. To add a matched Nef open reading frame, the stop codon in polwas removed, and synthetic clade A nef/h (GenBank accession number:AF069670) was fused to the 3′ end of pol/h by PCR to generate the cladeA plasmid, pVRC-4313. For the clade B Gag-Pol-Nef fusion protein,sequence encoding amino acids 1 to 432 from a CCR5-tropic clade B(GenBank accession number K03455) was used and fused to the pol/hdescribed above. To add a clade B Nef protein, the stop codon from Polwas removed and fused to a clade B synthetic Nef/h gene (aa 1 to 206)from HIV-1 PV22 (GenBank accession number K02083) to generate the cladeB plasmid, pVRC-4306. For the clade C Gag-Pol-Nef fusion protein, aminoacids 1 to 432 from a CCR5-tropic clade C (GenBank accession numberU52953) were used and fused to the pol/h gene described above. The Polstop codon was removed and fused to synthetic clade C Nef/h (aa 1 to206) (GenBank accession number: U52953), designated pVRC-4311.

Alternative Clade Env Plasmid DNAs. The sequences used to create the DNAplasmids encoding Env are derived from three HIV-1 CCR5-tropic strainsof virus that have been modified to reduce potential cellular toxicityand increase immunogenicity by deletion of the fusion domain, thecleavage domains, and also by shortening of the interspace betweenheptad 1 (H1) and heptad 2 (H2), as described previously for clade βisolates (Huang, Y. et al. 2001 J Virol 75:4947-4951). The syntheticprotein sequence for the clade A Env polyprotein (gp160) was derivedfrom 92rw020 (R5-tropic, GenBank accession number U51283) and designatedclade A gp145ΔCFI/h. An XbaI site was inserted 18 nucleotides upstreamfrom the ATG, together with a known Kozak sequence, and a BamH1 sitecreated 1,912 nt downstream of the ATG for all Env expression vectors.This fragment was cloned into the XbaI-to-BamH1 sites of pVR1012x/ssites. The fusion and cleavage domains from amino acids 486-519 and theinterspace between H1 and 1-12 from amino acids 576-604 were deleted.The protein sequence of the clade B Env glycoprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) by alteration of codons forbetter expression in human cells. The nucleotide sequence X4gp160/hshows little homology to the HXB2 gene, but the protein encoded is thesame with the following aa substitutions: aa 53 (Phe→Leu), aa 94(Asn→Asp), aa 192 (Lys→Ser), aa 215 (Ile→Asn), aa 224 (Ala→Thr), aa 346(Ala→Asp), and aa 470 (Pro→Leu). To produce an R5-tropic version of theenvelope glycoprotein (R5gp160/h), the region encoding HIV-1 envelopeglycoprotein amino acids 205 to 361 from X4gp160/h was replaced with thecorresponding region from the BaL strain of HIV-1 (GenBank accessionnumber M68893, again using human-preferred codons). The full-lengthCCR5-tropic version of the envelope gene from pR5gp160/h was terminatedafter the codon for aa 704 to generate gp145/h. The fusion and cleavagedomains from amino acids 503-536 and the interspace between H1 and H2from amino acids 593-620 were then deleted. The protein sequence of theclade C Env polyprotein (gp145ΔCFI) from 97ZA012 (R5-tropic, GenBankaccession number AF286227) was used to create a synthetic version of thegene (clade C gp145ΔCFI/h) with deletion of the fusion and cleavagedomains from amino acids 487-520 and the interspace between H1 and H2from amino acids 577-605.

Immunizations. Mice received two 100-μl injections intramuscularly ineach thigh at days 0, 14 and 42. Ten days after the final injection,mice were bled and sera were collected. Then the mice were sacrificed,spleens were removed, and the spleen cells were analyzed byintracellular cytokine flow cytometry (ICC) for CD4+ and CD8+ T-cellresponses.

Flow Cytometric Analysis of Intracellular Cytokines. CD4+− and CD8+−T-cell responses were evaluated by using intracellular cytokine flowcytometry (ICC) for gamma interferon (IFN-γ) and tumor necrosisfactor-alpha (TNF-α). This sensitive assay was developed to study theimmune responses to HIV-1 (Dorrell, L. et al. 2001 Eur J Immunol31:1747-1756; Goepfert, P. A. et al. 2000 J Virol 74:10249-10255;Maecker, H. T. et al. 2001 J Immunol Methods 255:27-40; Migueles, S. A.& Connors, M. 2001 Immunol Lett 79:141-150; Novitsky, V. et al. 2001 JVirol 75:9210-9228). The assay was performed by removal of spleens,gentle homogenization to single-cell suspension, erythrocyte lysis withPharMLyse (BD-Pharmingen), washing with medium, and stimulation (107cells/ml) at 37° C. for 1 h with peptide pools (2.5 μg/ml for eachpeptide). All peptides used in this study were 15-mers overlapping by 11amino acids that spanned the complete sequence of the HIV or negativecontrol proteins tested. Anti-CD28 and anti-CD49d antibodies(BD-Pharmingen 553294 and 553153 respectively) were added (1 μg/ml) tothe medium for costimulation. After an hour, brefeldin A (Sigma) wasadded to the medium (10 μg/ml) for an additional 5 h. After a total of 6h, cells were washed and incubated with FC block (BD-Pharmingen) for 15min on ice, fixed, and permeabilized with Cytofix/Cytoperm(BD-Pharmingen) according to manufacturer's instructions. The cells werewashed with phosphate-buffered saline (PBS) with 0.1% saponin (Sigma)followed by staining with the indicated fluorescent-labeled monoclonalantibodies against CD3, CD4, CD8, IFN-γ and TNF-α (BD-Pharmingen) for 20min on ice. After washing with PBS with 0.1% saponin, the cells wereanalyzed by fluorescence-activated cell sorting (FACS) to detect theIFN-γ and TNF-α-positive cells in the CD4+ and CD8+ cell populations andanalyzed with the program FlowJo (Tree Star, Inc.).

ELISA Assays. To detect antibodies against Env proteins of differentclades, enzyme-linked immunosorbent assay (ELISA) plates were coatedwith 100 μl of Galanthus Nivalis lectin (10 μg/ml) overnight at 4° C.The lectin solution was removed from the wells and blocked with 200 μlof PBS containing 10% fetal bovine serum (FBS) for 2 h at roomtemperature. The plates were washed twice with PBS containing 0.2%TWEEN™ 20 (PBS-T) and then 100 μl of supernatant from cells transfectedwith pVRC5304 (R5 gp140ΔCFI-Clade-A), pVRC2801 (R5 gp140ΔCFI-Clade-B),or pVRC5308 (R5 gp140ΔCFI-Clade-C) was added to each well, and wellswere incubated for an hour at room temperature. The plates were washedwith PBS-T five times, and then the sera from immunized mice fromdifferent groups were added with 3-fold dilutions for 1 h. The plateswere washed with PBS-T five times, and then 100 μl of 1:5,000-dilutedsecondary antibody-conjugated horseradish peroxidase (HRP) was added,and mixtures were incubated for 1 h, and washed with PBS-T five times.Then 100 μl of substrate (Sigma Fast o-phenylenediamine dihydrochloride,catalog #P-9187) was added in each well for 30 min. The reaction wasthen stopped by adding 100 μl of 1 N H2SO4 and the optical density (OD)reading was taken at 450 nm.

Statistical Analysis. For the simpler combination of plasmids listed inTable 1, Kruskal-Wallis tests were performed to test for overalldifferences in the three treatment groups' CD4+ and CD8+ response rateswithin each gene and clade combination at an α of 0.05. Within each ofthe two sets of tests (CD4+ and CD8+ responses), the Holm procedure wasused to adjust the P values for multiple comparisons for each gene andclade combination. If the adjusted P value from the Kruskal-Wallis testfor a given response-gene-clade combination was less than an α of 0.05,two-sided Wilcoxon tests were performed for all three possible pairs ofdifferent combinations (control vs. ABC(×4), control vs. ABC(×6),ABC(×4) vs. ABC(×6)). Again, the Holm procedure was used to adjust the Pvalues for multiple comparisons. An adjusted P value less than an α of0.05 was taken as evidence of a significant difference. An analogousapproach was taken to test for differences among the groups immunizedwith Env and Gag-Pol-Nef plasmids (FIG. 8).

Results

A combination of Env and Gag-Pol-Nef plasmids elicited CD4⁺ and CD8⁺responses to Env and Gag similar to those obtained with single plasmidsalone. To examine whether combined immunization with Env and Gag-Pol-Nefplasmids would enhance or inhibit antigen-specific responses, the CD4,CD8, and antibody responses to Env were analyzed. Four groups of micewith 5 mice per group were immunized with the control vector alone, Envwith control vector as filler DNA, Gag-Pol-Nef with control vector asfiller DNA, or Env with Gag-Pol-Nef. Ten days after the final DNAimmunization, animals were sacrificed, and splenocytes were incubatedwith overlapping Gag peptide pools. Intracellular IFN-γ and TNF-αexpression in stimulated CD4+ or CD8+ lymphocytes were analyzed by flowcytometry, and positive cells were enumerated. Cells from mice immunizedwith Gag-Pol-Nef alone and those immunized with the combination of Envand Gag-Pol-Nef responded similarly to Gag stimulation (FIG. 8A, left).Likewise, lymphocytes from mice vaccinated with Env alone, and thosewith a combination of Env and Gag-Pol-Nef, responded similarly toincubation with Env peptide pools (FIG. 8A, right). Based on statisticalanalysis, there was no difference in CD4 response to Gag between theGag-Pol-Nef group and the combined Env and Gag-Pol-Nef group (P=0.1746).Also, there was no difference in the CD4 response to Env between the Envgroup and the combined Env and Gag-Pol-Nef group (P=0.6905). In the caseof CD8 responses to Gag, there was also no statistical differencebetween Gag and the combined Env and Gag-Pol-Nef group (P=1.0), and inthe case of the CD8 responses to Env, there was also no statisticaldifference between Env and the combined Env and Gag-Pol-Nef group(P=1.0). Similarly, antibody to Env showed similar titers in both groups(FIG. 8B). There was no statistical difference between Env and thecombined Env and Gag-Pol-Nef group (P>0.05) in antibody response to Envat all four dilutions. This result indicated that combination plasmidvaccination did not cause immune interference but instead led toexpanded breadth of the immune response. To deteiiuine whether theaddition of alternative clades would prove similarly immunogenic, morecomplex plasmid combinations were evaluated.

Combination of Env clades and Gag-Pol-Nef vaccination elicits similarimmune responses to single clade immunogens. We next determined whetherthe inclusion of multiple Env immunogens would affect the breadth andpotency of the immune response. Mice were immunized with a negativecontrol plasmid, combined Env and Gag-Pol-Nef (both from clade B), orEnv from clades A, B and C with Gag-Pol-Nef from clade B, termedABC(×4). In the ABC(×4) group, the three Env proteins were retained inequal proportions, and the ratio of all Env proteins to all Gag-Pol-Nefproteins was kept constant (1:1, wt/wt). Both the combined Env andGag-Pol-Nef group and the ABC(×4) group induced CD4+ and CD8+ responsessimilar to those obtained with clade B Env (FIG. 9A). Some minorvariations in immune responses were seen between groups; however, boththe clade B and the ABC(×4) groups showed comparable CD4+ and CD8+responses to clade B Env peptide stimulation by intracellular flowanalysis. For antibody responses, ABC(×4) showed a measurable responseto clade A Env stimulation but as expected, not in the clade B-immunizedgroup, which did not contain Clade A Env. More importantly, immunizationwith the clade B immunogens gave rise to titer responses to clade B Envsimilar to those obtained with ABC(×4), again showing that the mixtureof multiple clades did not inhibit the responses to a single-clade (B)Env component, despite the relative dilution of the immunogen. Neitherthe ABC(×4) nor the clade C Env alone induced a high-titer antibodyresponse, possibly because of the lack of highly reactive epitopes inmice (FIG. 9B). These results indicated that the addition of multipleEnv proteins from alternative clades to Gag-Pol-Nef did not interferewith T-cell or humoral immunity and instead added breadth to the immuneresponse.

Comparison of different combination multiple clade immunogens. We nextcompared different combinations of plasmids that could elicit immuneresponses to multiple immunogens. Mice were immunized with the controlplasmid and two combinations of plasmids (Table 1), including acombination of six plasmids, designated ABC(×6), because it covered theGag, Nef, and Env from Clades A, B and C with Pol from Clade B, or theABC group with four components, ABC(×4), in which the Gag-Pol-Nef fusionprotein from Clade B was used alone, rather than with the Gag-Pol-Nefproteins from Clades A and C. As above, the three Env clades wereretained in similar ratios and amounts in both formulations, and theratio of all Env proteins to all Gag-Pol-Nef proteins was kept constant(1:1, wt/wt).

TABLE 1 Experiment schema for analysis of plasmid combinations in mice.Design of study to test different combinations of plasmids, with 10 miceper group. Vaccine Plasmid Amount VR1012 1012  50 μg ABCx41012-A-gp145ΔCFI 8.3 μg 1012-B-gp145ΔCFI 8.3 μg 1012-C-gp145ΔCFI 8.3 μg1012-B-gag-pol-nef  25 μg ABCx6 1012-A-gp145ΔCFI 8.3 μg 1012-B-gp145ΔCFI8.3 μg 1012-C-gp145ΔCFI 8.3 μg 1012-A-gag-pol-nef 8.3 μg1012-B-gag-pol-nef 8.3 μg 1012-C-gag-pol-nef 8.3 μg

Both plasmid combination groups had similar CD8 response to Gag, Pol,and Env from Clade B, but not from other clades (FIG. 10) and Table 2.Responses to Gag from Clades A and B were significantly higher than thecontrol (pVR1012) for both ABC(×6) and ABC(×4), but the differencesbetween the response rates of the two treatment groups were notsignificantly different for either of these clades. CD8+ responses toPoh-1 and Env from Glade B were significantly higher than the control(pVR1012) responses for both ABC(×6) and ABC(×4). However, CD8+responses to Env from Clade A were higher than the control for ABC(×6)only (P=0.0316) (FIG. 10C) and Table 2.

TABLE 2 Summary of the T cell and antibody responses to differentvaccine candidates. Response to: A- B- C- A- B- C- B- B- A- B- C-Analysis Vaccine gag gag gag env env env pol- pol- nef nef nef ICC^(a)ABC(x4) + + − + + + + + − + − CD4 ABC(x6) + + + + + ++ − + − − − ICCABC(x4) + + − − + − + − − − − CD8 ABC(x6) + + + + + − + − − − −ELISA^(b) ABC(x4) + + + ABC(x6) + + + ^(a)CD4⁺- and CD8⁺-T-cellresponses to different vaccine candidates. When Holm-adjustedKruskal-Wallis tests indicated overall significant differences, the datafrom all possible pairs of groups were compared by Wilcoxon tests with aHolm adjustment for the multiple comparisons. −, no statisticallysignificant difference from the control (P > 0.05); +, statisticallysignificant difference from the control only (P < 0.05); ++,statistically significant difference from both the control and all othertreatment groups (P = 0.05). ^(b)Antibody responses to different vaccinecandidates. +, the average antibody titer of the group was more than1:1,000

ABC(×6) and ABC(×4) induced similar CD4+ responses, in contrast to thecontrol (pVR1012) plasmids in mice. Both stimulated higher CD4-responsesfor Gag from Clade A and B, Pol-2 from Clade B, and Env from Clades Aand B (Table 2). ABC(×6) elicited significantly higher CD4+ responses toclade C Gag (P=0.0138) than the control (pVR1012), while for Nef andPol-1 from Clade B, only ABC(×4) provoked significantly higher CD4+responses than the control (P=0.0097 for Nef, P=0.0054 for Pol-1). CD4+responses to Env from Clade C were higher for ABC(×6) compared toABC(×4) (P=0.0418), although the responses for both groups weresignificantly higher than the control (pVR1012) (FIG. 11 and Table 2).ABC(×4), but not ABC(×6), showed a response to Nef from Clade B (Table2). In summary, except for relatively minor differences in specificity,both ABC(×6) and ABC(×4) elicited comparable cell-mediated immuneresponses.

Similar antibody responses in ABC(×4), ABC(×6) and single cladevaccinated mice to Envs from all clades. Sera from ABC(×4), ABC(×6), orsingle-clade groups were tested for antibody responses using alectin-capture HIV-1 Env protein ELISA system. The sera from the twotest groups showed similar responses to Env protein to all three clades(FIG. 11B). Antibody titers against Clade A Env protein from bothABC(×4) and ABC(×6) groups were higher than the antibody titers againstClade B and Clade C Env protein; however, there was no significantdifference between the two groups in terms of their antibody responsemagnitude. This result indicated that addition of Gag and Nef immunogensfrom Clade A and Clade C to ABC(×4) groups did not interfere with theantibody responses against Env from Clade B.

Discussion

One requirement of a highly effective AIDS vaccine is the need to induceboth neutralizing antibodies and cellular immunity to the many strainsof HIV-1 that circulate throughout the world. In this study, we haveevaluated the ability of plasmid DNA vaccines to elicit immune responsesto multiple gene products of HIV-1 from alternative clades of virus. Thegoal was to elicit both antibody and T cell responses against variousHIV genes from these different clades. Env, Gag, Pol and Nef were chosenas targets because they represent the major expressed proteins duringviral infection. A mutant Env with deletions in the cleavage site,fusion domain, and a region between the heptad repeats was used for itsability to elicit a more potent humoral immune response while retainingits ability to stimulate Env-specific cytotoxic T lymphocytes (CTL)(Chakrabarti, B. K. et al. 2002 J Virol 76:5357-5368).

A variety of previous studies have shown that CTL contribute to thecontrol of viremia and protect against the progression of HIV disease(Borrow, P. et al. 1994 Virol 68:6103-6110; Jin, X. et al. 1999 J ExpMed 189:991-998; Klein, M. R. et al. 1995 J Exp Med 181:1365-1372; Koup,R. A. et al. 1994 J Virol 68:4650-4655; Moss, P.A. H et al. 1995 PNASUSA 92:5773-5777; Musey, L. et al. 1997 N Engl J Med 337:1267-1274; Ogg,G. S. et al. 1998 Science 279:2103-2106; Rowland-Jones, S. L. et al.1998 J Clin Invest 102:1758-1765; Rowland-Jones, S. L. et al. 1993Lancet 341:860-861; Rowland-Jones, S. L. et al. 1995 Nat Med 1:59-64;Schmitz, J. E. et al. 1999 Science 283:857-860). Processed forms of Gag,Pol, Nef and Env presented on class I MHC antigens can serve as thetargets of CTL that recognize and lyse HIV-1 infected cells, in this waycontributing to the efficacy of a preventive vaccine. If the T cellresponse is sufficiently robust, it is hoped that these cells will killHIV-infected cells before the virus can replicate and establish areservoir of infection in vivo. For a globally effective vaccine, itwill be necessary to elicit CTL that react with strains from multipleclades. Though there may be some cross-clade reactivity afterimmunization with a single clade (e.g., Keating, S. M. et al. 2002 AIDSRes Hum Retroviruses 18:1067-1079), there is also evidence ofdisparities in such immune responses, (e.g., Dorrell, L. et al. 2001 EurJ Immunol 31:1747-1756). It therefore is desirable to includerepresentatives of the major classes of virus in a DNA vaccine to inducecross-clade immunity. However, the main concern of such a cocktail iswhether it will cause interference between gene-specific immuneresponses. Interference among immune responses to various viral geneshas been seen previously in murine HIV immunization studies (Kjerrstrom,A. et al. 2001 Virology 284:46-61; Muthumani, K. et al. 2002 Vaccine20:1999-2003). Recently, studies of modifications to HIV DNA vaccines,including different combinations of viral genes, altered RNAstructure/codon usage, and/or stimulatory cytokine genes, have shownmore encouraging results in mice (zur Megede, J. et al. 2003 J Virol77:6197-6207). More importantly, some approaches have shown promise inchallenge studies using non-human primates (Amara, R. R. et al. 2001Science 292:69-74; Barouch, D. H. et al. 2000 Science 290:486-492; Kim,J. J. et al. 2001 Virology 285:204-217; Letvin, N. L. 2002 J Clin Invest110:15-20; McKay, P. F. et al. 2002 J Immunol 168:332-337; Shiver, J. Wet al. 2002 Nature 415:331-335), though complete protection againstinfection has been difficult to achieve. Additional modifications weretherefore incorporated in this study in an attempt to improve efficacy.

When the immune responses to different combinations of Env andGag-Pol-Nef were compared, there was no decrease in the humoral andcellular response to Clade B mutant Env plasmid and Gag-Pol-Nef plasmidswhen mixed compared with the two plasmids individually (FIG. 8). Whenthe complexity was increased to four components, including gp145ΔCFIfrom three clades and Gag-Pol-Nef from Clade B, there was nointerference with the humoral response to B-env at the same time thatthe immune response to other clades was enhanced.

When the complexity of the vaccine was increased to six components,ABC(×6), containing the same Env-gp145ΔCFI from different clades as ingroup ABC(×4) plus the Gag-Pol-Nef fusion protein from clades A and C,minor differences in immunogenicity were seen. Analyzing the Gagresponse, ABC(×4) elicited CD4⁺ and CD8⁺ responses to clades A and B,while ABC(×6) improved the response to clade C Gag peptides. The lack ofCD4⁺ and CD8⁺ responses to Clade C Gag in ABC(×4) probably is due to theabsence of clades A and C Gag; however, ABC(×4) containing only clade BGag could induce both CD4⁺ and CD8⁺ responses to clade A Gag even thoughit shares only 85% homology in amino acid sequence. This resultindicated that clades A and B Gag share some common CD4⁺ and CD8⁺epitopes but differ more substantially from clade C Gag in mice. Incontrast, the CD4⁺ and CD8⁺ responses against Env between ABC(×4) andABC(×6) were similar: both groups elicited comparable CD4⁺ responsesagainst all three clades and generated similar CD8⁺ responses againstclade B Env. ABC(×6) also induced a significant CD8⁺ response to Envfrom Clade A.

For Pol responses, both groups demonstrated CD4⁺ and CD8⁺ responsesagainst Pol from Clade B. The ABC(×4) group elicited a CD4⁺ response toboth sets of Pol peptides, while ABC(×6) stimulated CD4⁺ response onlyagainst one of the two Pol peptide pools (Table 2). Both groups inducedCD8⁺ responses to the first half of the Clade B poi (FIG. 10B, leftpanel). For Nef, only the ABC(×4) group elicited a CD4+ response againstNef from clade B (Table 2). The poor anti-Nef response also may be dueto the inability of Balb/c mice to recognize Nef epitopes, as othergroups have reported that Nef is highly immunogenic in other strains ofmice (Kjerrstrom, A. et al. 2001 Virology 284:46-61).

In addition, we attempted to determine whether CD4⁺ and CD8^(±)T cellresponses against multigenes would affect humoral responses. There wasno significant difference among different groups in ELISA titers(summarized in Table 2). All the groups showed similar antibody titersto Env protein from Clades A, B and C (FIG. 11B). These data indicatedthat there was no interference among different clades of Env in antibodyresponse. Equally importantly, there was no interference among variousviral genes between humoral and cellular responses.

In summary, the ABC(×4) vaccine regimen was able to induce substantialand balanced CD4⁺ and CD8⁺ T cell responses to the viral antigens fromdifferent clades. The results here indicate that a multi-gene HIV-1 DNAvaccine is feasible because the immune responses to individual genes donot cause interference when combined with one another. As the HIV-1pandemic continues to grow, the concern about virus variability becomesincreasingly problematic. Though a few subtypes of HIV-1 predominate indifferent regions of the world, a rising number of recombinant strainshave been reported lately (Kuiken, C. et al. 2000. Human Retrovirusesand AIDS 1999. Los Alamos National Laboratory, Los Alamos, N. Mex.).Such viruses continually mutate and escape (Barouch, D. H. et al. 2002Nature 415:335-339; Mortara, L. et al. 1998 J Virol 72:1403-1410) duringdifferent stages of infection. A multiepitope and multiclade immuneresponse should help to reduce the likelihood of viral escape. The datapresented in this study therefore guides the development of improvedvaccines against diverse strains of HIV.

Part III Selective Modification of Variable Loops Alters Tropism andEnhances Immunogenicity of HIV-1 Envelope

Abstract

Although the B clade of HIV-1 envelope (Env) includes five highlyvariable regions, each of these domains contains a subset of sequencesthat remain conserved. The V3 loop has been much studied for its abilityto elicit neutralizing antibodies, which are often restricted to alimited number of closely related strains, likely because a large numberof antigenic structures are generated from the diverse amino acidsequences in this region. Despite these strain-specific determinants,subregions of V3 are highly conserved, and the effect of differentportions of the V3 loop on Env tropism and immunogenicity has not beenwell delineated. In this study, selective deletions in V3 have beenintroduced by shortening the stem of the V3 loop. These mutations wereexplored in combination with deletions of selected V regions.Progressive shortening of the stem of V3 abolished the immunogenicity aswell as the functional activity of HIV Env; however, two small deletionson both arms of the V3 stem altered the tropism of the dual-tropic 89.6Pviral strain so that it infected only CXCR4⁺ cells. When this smallerdeletion was combined with removal of the V1 and V2 loops and used as animmunogen in guinea pigs, the antisera were able to neutralize multipleindependent clade B isolates with higher potency. These findingsindicate that highly conserved subregions within V3 are relevant targetsto elicit neutralizing antibody responses, affecting HIV tropism, andincreasing the immunogenicity of AIDS vaccines.

Introduction

Among the mechanisms used by HIV to avoid immune recognition andantibody neutralization, the variable regions of the envelope play animportant role in evasion. The envelope protein utilizes a variety ofmechanisms to evade detection, including carbohydrate modification,conformational flexibility, and genetic variability between isolates(Burns, D. P. & Desrosiers, R. C. 1994 Curr Top Microbiol Immunol188:185-219; Burton, D. R. 2002 Nat Rev Immunol 2:706-713; Chakrabarti,B. K. et al. 2002 J Virol 76:5357-5368; Gorny, M. K. et al. 2002 J Virol76:9035-9045; Kwong, P. D. et al. 2002 Nature 420:678-682; Wei, X. etal. 2003 Nature 422:307-312). Genetic diversity in specific segments ofthe viral Env protein gives rise to the variable regions. These regionsserve to block access to the CD4 binding domain as well as the chemokinereceptor binding site, in addition to influencing virus neutralizationsensitivity and being responsible for strain specificity of neutralizingantibodies (Guillon, C. et al. 2002 J Virol 76:2827-2834; Parren, P. W.H. I. et al. 1999 AIDS 13:S137-S162; Wyatt, R. & Sodroski, J. 1998Science 280:1884-1888). Although the variable regions have been definedby their genetic differences among alternative isolates, it is clearthat there are subregions within the V loops that show some degree ofconservation. This sequence homology is particularly evident in suchregions as the tip of the V3 loop (Korber, B. T. et al. 1994 J Virol68:7467-7481). Other motifs can also be identified in various virusstrains. For example, specific N-linked glycosylation sites andsequences near the base of the V3 loop are well conserved (Korber, B. T.et al. 1994 J Virol 68:7467-7481). In this study, the fine specificityof the variable regions was explored in further detail. Specifically,the V3 loop has been examined with regard to the contribution of theputative stem structures to viral tropism and immunogenicity. We foundthat a specific mutation that shortens the stem of the V3 loop can alterthe tropism of HIV envelope. This mutation, in combination with deletionof the V1 and V2 loops, further enhances the ability of the envelope toelicit a neutralizing antibody response.

Materials And Methods

Antibody. Anti-HIV-1 human monoclonal antibody 2F5 (Purtscher, M. et al.1996 AIDS 10:587-593) and human HIV immunoglobulin G (IgG) were obtainedfrom the National Institutes of Health (NIH) AIDS Research and ReferenceReagent Program, Division of AIDS, NIAID, NIH. Anti-HIV p24 antibodyKC57-RD1 was obtained from Beckman Coulter, Inc.

Cell and virus stocks. Human embryonic kidney cell 293 was purchasedfrom ATCC, and maintained in Dulbecco's modified Eagle's media(Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum (FBS)and 100 μg/ml of penicillin/streptomycin. The human T-cell leukemia cellline MT-2 and the HeLa-derived cell line MAGI-CCR5 were obtained fromthe AIDS Research and Reference Reagent Program, Division of AIDS,NIAID, NIH.

HIV-1 isolates (ADA, JRCSF, JRFL, Bal, SF162 and 89.6) were obtainedfrom the NIH AIDS Research and Reference Reagent Program, Division ofAIDS, NIAID, NIH. Primary isolates 6101 (previously called P15) and 1168were obtained from David Montefiori of Duke University (Bures, R. et al.2000 AIDS Res Hum Retroviruses 16:2019-2035). The viruses were expandedby two or three cycles of growth on phytohemagglutinin (PHA)- andinterleukin (IL-2)-stimulated peripheral blood mononuclear cells (PBMC).For the production of the working stock virus, PBMC were exposed toundiluted virus for 2 h at a concentration of 10⁷ cells/ml. IL-2 culturemedium was added to bring the concentration to 10⁶ cells/ml. The IL-2culture medium was changed every 2 days, and supernatants were collectedduring the peak of p24 expression, usually 5-10 days after infection.Virus stocks were made cell free centrifugation at 1,000×g andfiltration through a 0.45-μm-pore-size filter. In some cases, viralstocks were concentrated by as much as 10-fold using a 100-kDa cutoffpolyethersulfone filter (Centricon Plus Biomax filter, Millipore,Bedford, Mass.), according to manufacturer's instructions. Virusaliquots were stored in the vapor phase of liquid nitrogen. Viruses BL01and BR07 were provided by Dana Gabuzda of the Dana-Farber CancerInstitute. Both are chimeric infectious molecular clones of HIV strainNL4-3 that contain the full-length env genes from primary HIV-1 isolates(Ohagen, A. et al. 2003 J Virol 77:12336-12345). After initial plasmidtransfection of 293 cells, these viruses were expanded in PBMC asdescribed above.

Buoyant density gradient analysis of lentiviral vectors. 293T cells(3×10⁶) were transfected with 3 μg each of the relevant Gag and Envexpression vectors in a 100-mm-diameter tissue culture dish withDulbecco's modified Eagle's medium. Three days later, the cellsupernatants were collected and mixed with 60% OptiPrep (iodixanol)medium (Invitrogen); the final concentration of OptiPrep was adjusted toa 30% density gradient formed by centrifugation at 45,000×g for 6 h in aVTI50 rotor (used according to the manufacturer's instructions;Invitrogen); and each fraction was collected according to the indicateddensity. Lentiviral vector proteins were separated in a sodium dodecylsulphate-4 to 15% polyacrylamide gel electrophoresis (SDS-4 to 15% PAGE)gel, transferred onto an Immobilon-P membrane, and plotted for theexpression of Gag (human HIV IgG, used at 1:5,000) and Env (human HIVIgG, used at 1:5,000).

Construction of recombinant adenoviruses. Adenovirus type 5 (Ad5)-basedfirst-generation (ΔE1, ΔE3) recombinant adenoviruses expressingdifferent V loop deletions of gp140(ΔCFI) were constructed as describedpreviously (Aoki, K. et al. 1999 Mol Med 5:224-231). In brief,PacI-linearized shuttle vectors containing V loop deletions ofgp140(ΔCFI) were recombined with the right side of Ad5 genomic DNAcarried in cosmid by use of Cre recombinase (Novagen, Madison, Wis.).The resulting recombinants were ethanol precipitated, dissolved inTris-EDTA, and transfected into 293 cells. Recombinant adenoviruses wereobserved based on plaque formation 10 to 14 days after transfection.Viruses were amplified, purified two times through a CsCl gradient, andstored in PBS+15% glycerol at −20° C.

Production of pseudotyped lentivirus. HIV-Luc pseudotyped with HIVgp160(89.6P) and its V3 deletion mutants were prepared according topublished methods (Naldini, L. et al. 1996 Science 272:263-267).Briefly, the packaging vector pMD 8.2, pHR-Luciferase, and theenvelope-expressing vector were transiently cotransfected into 293Tcells by use of calcium phosphate. Supernatants were harvested 48 and 72h after transfection, filtered, and stored at −80° C. Virusconcentrations were determined by an ELISA assay for the p24 antigen(Coulter). The same amount of virus was added onto MT-2 (X4 tropic) andMAGI-CCR5 (R5 tropic) cells, and the cells were incubated for 2 h at 37°C. The cells were harvested 48 h after infection and lysed in cellculture lysis buffer (Promega, Madison, Wis.). The luciferase assay wasperformed according to the manufacturer's recommendation (Promega,Madison, Wis.).

Plasmid construction. Plasmid pVRC1012-gp140(ΔCFI) (HXB2/BaL chimera)and pVRC1012-gp145(ΔCFI) (HXB2/BaL chimera) have been describedpreviously (Chakrabarti B K et al. 2002 J Virol 76:5357-5368). To makegp140(ΔCFI)(ΔV₁V₂) and gp145(ΔCFI)(ΔV₁V₂), PCR was performed to amplifyan Xbal/NheI fragment covering ATG and the boundary of V1 loop usingprimers 5′CCTCTAGACACCATGCGCGTGAAGGAGAAG3′ (SEQ ID NO: 15) and5′CCGCTAGCGTCGGTGCACTTCAGGCTCACGCACAGGGG3′ (SEQ ID NO: 16) and anNheI/ApaI fragment covering the 3′ boundary of the V2 loop and the C3region using primers 5′CCGCTAGCACCAGCTGCAACACCAGCGTGATCACCCAG3′ (SEQ IDNO: 17) and 5′GGTGCAGGGGCCCTTGCCGTTGAACTTCTT3′ (SEQ ID NO: 18). TheXbaI/NheI- and NheI/ApaI-digested PCR fragments were cloned intoXbaI/ApaI-digested pVRC1012-gp140(ΔCFI) and pVRC1012-gp145(ΔCFI). Theresulting plasmids pVRC1012-gp140(ΔV₁V₂) and pVRC1012-gp145(ΔCFI)(ΔV₁V₂)have deletions of the V1 and V2 loops as follows: CTDASTSC (SEQ ID NO:19). Two extra amino acids (AS) were introduced due to introduction ofNheI site. A similar approach was used to make other V loop deletionmutants of gp145DCFI (HXB2/BaL chimera) and gp140ΔCH (HXB2/BaL chimera).The amino acid sequences of deleted V loops are as follows: ΔV₃,CTDASKNC (SEQ ID NO: 34); ΔV₂, CSFASTSC (SEQ ID NO: 35); ΔV₃, CTRASAHC(SEQ ID NO: 36); and ΔV₄, CNSASLPC (SEQ ID NO: 37).

V3 deletion mutants were made using the PCR-based Quickchange(Stratagene, La Jolla, Calif.) method according to the manufacturer'sinstructions. Each mutant was confirmed by double strain sequencing. AnApaI/SexAI fragment containing each confirmed V3 deletion was swappedwith a corresponding fragment in pVRC1012-gp140(ΔCFI)(ΔV₁V₂) andpVRC1012-gp145(ΔCFI)(ΔV₁V₂). The cDNA encoding gp160(89.6P)(KB9)(Karlsson, G. B. et al. 1997 J Virol 71:4218-4225) was synthesized byusing human preferred codons. Plasmids expressing different V3 deletionmutants of gp160(89.6P) were made similarly and are shown in FIG. 12.The details for each V3 mutant are listed in FIG. 13A.

ELISA assay. Guinea pig anti-HIV gp140(ΔCFI) ELISA titer was measured byusing a modified lectin capture method. Briefly, Immunon 2HB ELISAplates (Thermo Labsystems, Franklin, Mass.) were coated with 100 μl ofGalanthus Nivalis lectin (Sigma, St. Louis, Mo.) (10 μg/ml in PBS)/wellovernight at 4° C. The plates were blocked with 200 μl of PBS containing10% FBS for 2 h at room temperature, and washed twice with PBScontaining 0.2% TWEEN™-20 (PBS-T). One hundred microliters of tissueculture supernatant from pVRC 1012-gp140(ΔCFI)-transfected 293 cells wasadded in each well and incubated at room temperature for 1 h. The plateswere washed 5 times with PBS-T. One hundred microliter serial dilutionsof guinea pig immune serum in PBS containing 1% FBS were then added intriplicate and incubated for 1 h at room temperature. After five washeswith PBS-T, 100 μl of horseradish peroxidase (HRP)-conjugated F(ab)'2donkey anti-guinea pig IgG (1:5,000) (Jackson ImmunoResearchLaboratories, West Grove, Pa.) in PBS+1% FBS was added to each well, andincubated for 1 h at room temperature. The plates were washed 5 timeswith PBS-T, developed by the addition of 100 μl of o-phenylenediaminedihydrochloride (Sigma, St. Louis, Mo.) (one gold and one silver tabletin 20 ml of water) and incubated at room temperature for 30 min. Thereaction was stopped by the addition of 100 μl of 1 NH₂SO₄ to each well.The readout was measured at 450 nm by a SPECTRAmax plate reader(Molecular Devices, Sunnyvale, Calif.). The endpoint dilution wascalculated by picking the dilution for which the readout was above thatof 1:100 dilution of preimmune serum.

Neutralization assay. The single-round intracellular p24-antigen flowcytometric HIV-1 neutralization assay has been described previously(Mascola, J. R. et al. 2002 J Virol 76:4810-4821). Briefly, 40 μl ofvirus stock was incubated with 10 μl of heat-inactivated guinea pigimmune serum (multiplicity of infection, approximately 0.1). Afterincubation for 30 min at 37° C., 20 μl of PBMC (1.5×10⁵ cells) was addedto each well. PBMC were maintained in IL-2 culture medium containing 1μM indinavir, and the cells were fed on day 1 with 150 μl of IL-2culture medium containing indinavir. One day after infection, cells werestained for intracellular p24-antigen with the KC57 anti-p24 antibody,followed by the quantitation of HIV-1 infected cells by flow cytometry.The percent of neutralization was defined as reduction in the number ofp24-positive cells compared with the number for wells incubated withcorresponding preimmune serum.

To obtain 50% inhibitory concentration (IC₅₀) and IC₈₀ data, serialdilutions of anti-serum were incubated with virus as described above.Antiserum dose-response curves were fit with a nonlinear function, andthe inhibitory dilutions that neutralized 50 and 80% (IC₅₀ and IC₈₀respectively) of virus were calculated by a least-squares regressionanalysis. Statistical analysis of IC₅₀ titers was performed using thenon-parametric Mann-Whitney rank-order test (GraphPad Prism softwarepackage V3.0, GraphPad Software Inc., San Diego, Calif.).

Vaccination. Guinea pigs were immunized intramuscularly with 500 μg (in400 μl PBS) of gp145 version of plasmid DNA at weeks 0, 2, and 6. Atweek 14, the guinea pigs were boosted with 10¹¹ (in 400 μl PBS)particles of recombinant adenovirus expressing the corresponding gp140version of the protein. Sera were collected at weeks—2 and 16, dividedinto aliquots, and frozen at −20° C.

Western Blotting. 293 cells were transfected with plasmid DNA expressingeach immunogen by the calcium phosphate method performed according tomanufacturer's instructions (Invitrogen). 48 h after transfection, thecells were harvested, washed once with PBS, resuspended in lysis buffer(50 mM HEPES pH 7.0, 150 mM NaCl, 1% NP-40, 1× proteinase inhibitorcocktail), and incubated on ice for 45 min. The cell lysate wascentrifuged at 14,000 rpm for 10 min at 4° C. The supernatant wascollected, and the protein concentration was measured. 20 μg of proteinwas mixed with 2× sample loading buffer (100 mM Tris, 4% SDS, 20%glycerol, 5% 2-mercaptoethanol, 0.2% bromophenol blue), and boiled for 5min. The sample was then resolved by 4 to 15% gradient SDS-PAGE andtransferred onto a nitrocellulose membrane (Bio-Rad, Hercules, Calif.).The membrane was blocked twice with Tris-buffered saline (TBS)containing 0.3 TWEEN™-20, 5% skim milk, and 1% bovene serum albumin(BSA) at room temperature for 10 min, followed by incubation with 2F5antibody (1:2,500) in blocking buffer for 1 h at room temperature. Themembrane was washed twice with 100 ml TBS containing 0.3% TWEEN™-20,followed by incubation with HRP-conjugated goat anti-human IgG(Chemicon, Temecula, Calif.) (1:5,000) for 30 min at room temperature.Following two washes with 100 ml of washing buffer, the membrane wasdeveloped using ECL Western blotting detection reagents (Amersham,Piscataway, N.J.), and exposed on Hyperfilm ECL (Amersham, Piscataway,N.J.).

Results

Expression of V region mutants. Modifications of three regions of theHIV envelope, the cleavage site, fusion peptide, and interhelicalcoiled-coil domain (ΔCFI) were shown previously to enhance the abilityof Env to elicit an antibody response (Chakrabarti, B. K. et al. 2002 JVirol 76:5357-5368). We evaluated additional mutations either indifferent V regions or through selective modifications of V3 (FIG. 12).The internal V3 loop deletions were made both in gp145ΔCFI (HXB2/BaLchimera), which was inserted into DNA expression vectors for primaryimmunization, and in gp140ΔCFI (HXB2/BaL chimera), which was placed intoan adenoviral vector for boosting. These series of mutations were alsointroduced into the strain 89.6P Env (FIG. 13A), a dual-tropic virusthat was analyzed initially in functional pseudotyping assays foreffects on tropism of different chemokine receptors. The expression ofthese progressive deletions of the V3 region was assessed by Westernblot analysis. Immunoreactive proteins of the expected molecular weightwere detected in cell lysates from 293T cells transfected with theseexpression vectors (FIG. 13B). These same mutations were also introducedinto the gp145ΔCFI (HXB2/BaL chimera) with V1 and V2 regions deleted,and protein expression was also confirmed (FIG. 13C).

Effects of V3 region mutations on Env function. To evaluate the effectsof progressive deletions in the V3 region, the 89.6P Env mutants wereanalyzed for their ability to mediate viral entry, using an HIV vectorencoding a luciferase reporter gene. The abilities of these V3 variantsto incorporate into pseudotyped lentivirus were confirmed by buoyantdensity centrifugation (FIG. 14A, B). Because this Env is dualtropic,both a CXCR4⁺ cell line, MT-2, and a CCR5⁺ indicator cell line,MAGI-CCR5, were tested. Although longer deletions of the V3 regionabolished the function of the 89.6 Env, the smallest deletion, whichremoves three amino acids on each side of the V3 loop, termed 1AB,preserved the ability of the Env to infect the CXCR4 target cell, MT-2(FIG. 14C, left panel). In contrast, even the smallest deletion of theV3 loop abolished its ability to infect MAGI-CCR5 cells (FIG. 14C, rightpanel). These data indicate that the length of the V3 loop or thedeleted amino acids play a critical role in determining its tropism foralternative chemokine receptors, and CCR5 tropism of this Env was moresensitive than CXCR4 tropism.

Effect of V region mutations on immunogenicity. To evaluate the effectof these and other V region mutations on the elicitation of aneutralizing antibody response, deletions of different V regions ingp145ΔCFI (HXB2/BaL chimera) and gp140ΔCFI (HXB2/BaL chimera),individually or with combinations of V1 to V4, were made. Expression ofthe mutants revealed similar levels of protein by Western blotting (FIG.15A). These V region mutants were assessed for their ability to elicitneutralizing antibodies using DNA/ADV immunization of guinea pigs. Theelimination of specific V regions, particularly the combination of V1and V3, or V1 and V4, markedly reduced their ability to induce aneutralizing antibody response. In contrast, vectors with specificcombined deletions, including V1 and V2 regions, increased theneutralizing antibody response to HIV^(BaL) (FIG. 15B). The increasedpotency of the V1V2 deletion construct was confirmed in furtherexperiments using nine additional primary HIV-1 isolates; these datastrongly indicated that this deletion construct provided betterimmunogenicity than the other constructs shown in FIG. 15M Based onthese analyses additional V3 region mutations were made in the V₁V₂deletion construct and the gp145ΔCFI envelope mutant. When testedagainst HIV^(BaL), gp145ΔCFI immunogen elicited slightly increasedneutralization compared to wild-type gp145, but it did not reachstatistical significance (FIGS. 15B and 16A). The less impressiveresponse for V₁V₂gp145ΔCFIΔ seen in FIG. 16A was due to a singlenonresponder in a group of four animals. The actual values in FIG. 16Afor gp145ΔCFI were 47, 60, 51, and 53, and those for V₁V₂ 145ΔCFIΔ were16, 71, 62, and 77%. However, we confirmed the improved immunogenicityof the ΔV₁V₂ mutant in numerous additional experiments. A furtherincrement was suggested when the 1AB mutation was included in theV₁V₂gp145ΔCFIΔ immunogen. When larger deletions of the V3 region weremade, they became successively less able to elicit a neutralizingantibody response. In contrast, all mutants were able to elicitcomparable antibody responses, as determined by ELISA end-point limitingdilution analysis (FIG. 16B).

Comparative neutralization profile of the V1V2 and V3(1AB) deletion. Toexamine the effects of these mutations on the breadth and potency ofneutralization, guinea pigs were immunized with selected mutants of thegp145 DNA followed by gp140 adenoviral vector boost, including thewild-type, ΔCFI, or ΔV₁V₂V₃(1AB)ΔCFI mutations. These modified Envs werecompared for their ability to elicit a neutralizing antibody response.For a comparison of potency of the neutralizing antibody response,serial dilutions of the guinea pig sera were tested against four primaryviruses (BaL, JRCSF, 89.6 and SF162), and the respective IC₅₀ valueswere calculated. Among the modified Env immunogens, the ΔV₁V₂V₃(1AB)ΔCFImutant was most effective in inhibiting these four isolates. Compared tothe wild-type, the median IC₅₀ of this construct was statisticallyhigher against two viruses (P=0.03 for JRCSF and 89.6) and was close tosignificance for one virus (P=0.06 for BaL) (FIG. 17A). Antiseraelicited by this optimal immunogen, ΔV₁V₂V₃(1AB)ΔCFI, were examinedagainst a panel of ten primary HIV-1 isolates. The antisera displayedreactivities against a number of unrelated HIV-1 strains. Five of theten viruses were moderately or strongly neutralized by a 1:5 dilution ofguinea pig sera. 50% neutralization was not achieved against threeviruses (ADA, 6101 and BL01), and two viruses were neutralized at a lowlevel (50%-60%) by one of the four guinea pig sera, therefore, theimmunogen remained limited in its breadth.

Discussion

In this study, we have examined the ability of different V-regionmutations to alter the immunogenicity of HIV envelope. We havepreviously shown that mutations in the cleavage site, fusion domain andinter-helical coiled-coil region can enhance immunogenicity by improvingthe ability of Env vaccines to elicit a neutralizing antibody response(Chakrabarti, B. K. et al. 2002 J Virol 76:5357-5368). Althoughimprovements were observed with the ΔCFI mutations in their ability toelicit antibody to Env, the enhancement in the neutralizing antibody wasless striking. The goal of the present study was therefore to expand thepotency and breadth of the neutralizing response by including additionalmodifications and systematically evaluating contributions of various Vregions and V3 subregions. Truncations in the V3 region markedly alteredthe functional properties of the HIV89.6P Env. Mutations exceeding sixamino acids, three each on opposite sides of the loop, abolishedfunction of both CXCR4− as well as CCR5− tropic viruses. In contrast,the smaller 1AB truncation eliminated the CCR5-tropic activity of thisenvelope but preserved its ability to target CXCR4⁺ cells. When the 1ABmutation was evaluated for its ability to elicit neutralizing antibodiesafter DNA priming and adenoviral vector boosting in guinea pigs, thismutation appeared to have the greatest efficacy in eliciting thisresponse. This effect required additional deletions of V1 and V2, as itwas not observed in the gp140/145ΔCFI background. After comparing thepotency of different immunogens, the breadth of the optimal candidatewas determined against ten representative clade B viral isolates. Thebreadth of this antisera was increased, with a higher IC₅₀ titer andincreased reactivity against different HIV isolates (FIG. 17).

Previous studies have indicated that subregions of V3 may be conservedamong various isolates and affect Env function. This conservation isevident in specific sequences in this region, for example, the tip ofthe V3 (Korber, B. T. et al. 1994 J Virol 68:7467-7481). Though the V3region has been shown to affect the tropism of HIV for the chemokinereceptor (Briggs, D. R. et al. 2000 AIDS 14:2937-2939), the effects ofprogressive deletions in the V3 loop and its selective effect on CXCR4targeting have not been previously appreciated. Recently, it has beensuggested that conserved conformational determinants are present in theV3 loop of diverse isolates that show similar sensitivity toneutralizing antibodies (Gorny, M. K. et al. 1997 J Immunol159:5114-5122; Krachmarov, C. P. et al. 2001 AIDS Res Hum Retroviruses17:1737-1748; Schreiber, M. et al. 1997 J Virol 71:9198-9205). Forexample, the ability of the monoclonal antibody 447-52D and related V3monoclonal antibodies to inhibit different strains with disparate V3sequences suggests that common determinants may be shared by geneticallydisparate strains (Gorny, M. K. et al. 1997 J Immunol 159:5114-5122; 8).The enhanced immunogenicity of the V1V2 mutations in this studyindicates that there may be masking of the V3 loop by V1 and V2 inHIVBal. Deletion of the V2 region has been suggested in previous studiesto improve the antibody response (Srivastava, I. K. et al. 2003 J Virol77:2310-2320), but it is not certain whether similar mechanisms areresponsible for those effects and the observations noted here in adifferent strain in combination with V3 partial deletions, since theadditional V1 deletion and the 1AB mutation in V3 further enhances itsimmunogenicity. The increased breadth of this response indicates thatcommon antigenic determinants are shared by many, though not all, cladeB viruses. Taken together, these conserved regions reflect underlyingfunctional requirements and structural homologies between differentviruses. Therefore, the families of V3 determinants are envisioned astargets for expansion of the breadth of the neutralizing antibodyresponse.

Part IV Heterologous Envelope Immunogens Contribute to AIDS VaccineProtection in Rhesus Monkeys

Abstract

Because a strategy to elicit broadly neutralizing anti-humanimmunodeficiency virus type 1 (HIV-1) antibodies has not yet been found,the role of an Env immunogen in HIV-1 vaccine candidates remainsundefined. We sought to determine whether an HIV-1 Env immunogengenetically disparate from the Env of the challenge virus can contributeto protective immunity. We vaccinated Indian-origin rhesus monkeys withGag-Pol-Nef immunogens, alone or in combination with Env immunogens thatwere either matched or mismatched with the challenge virus. Theseanimals were then challenged with a pathogenic simian-humanimmunodeficiency virus. The vaccine regimen included a plasmid DNA primeand replication-defective adenoviral vector boost. Vaccine regimens thatincluded the matched or mismatched Env immunogens conferred betterprotection against CD4⁺ T-lymphocyte loss than that seen with comparableregimens that did not include Env immunogens. This increment inprotective immunity was associated with anamnestic Env-specific cellularimmunity that developed in the early days following viral challenge.These data indicate that T-lymphocyte immunity to Env can broaden theprotective cellular immune response to HIV despite significant sequencediversity of the strains of the Env immunogens and can contribute toimmune protection in this AIDS vaccine model.

Introduction

The diversity of envelope (Env) proteins in human immunodeficiency virus(HIV) isolates worldwide poses a challenge for the development of aneffective AIDS vaccine. The failure of traditional vaccine strategies toprovide protection against HIV infection is attributable, at least inpart, to the genetic heterogeneity of Env (Letvin, N. L. et al. 2002Annu Rev Immunol 20:73-99). Env diversity underlies many of the problemsassociated with eliciting antibody responses that neutralize a varietyof HIV isolates (Mascola, J. R. 2003 Curr Mol Med 3:209-216). Thisdiversity also poses difficulties for generating T-lymphocyte responsesthrough vaccination that recognize genetically varied viruses (Letvin,N. L. et al. 2002 Annu Rev Immunol 20:73-99). In fact, the problemsassociated with Env diversity have raised questions about the utility ofincluding an Env immunogen in candidate HIV vaccines.

Nonhuman primates have been powerful models for evaluating HIV vaccinestrategies. Studies with macaques have provided evidence for thecritical contribution of cellular immunity in controlling AIDS virusreplication (Jin, X. et al. 1999 J Exp Med 189:991-998; Schmitz, J. E.et al. 1999 Science 283:857-860) and have illustrated the ability ofvaccines to modify the clinical course of disease even when suchvaccines cannot confer frank protection against infection with an AIDSvirus isolate (Barouch, D. H. et al. 2000 Science 290:486-492; Amara, R.R. et al. 2001 Science 292:69-74). Moreover, the rationale for advancinga number of vaccine modalities into early-phase human trials derivesfrom studies in nonhuman primates (Letvin, N. L. et al. 1997 PNAS USA94:9378-9383; Shiver, J. W. et al. 2002 Nature 415:331-335).

Recent studies with nonhuman primates have suggested thatvaccine-elicited Env-specific immune responses can contribute tocontainment of simian immunodeficiency virus (SIV) and simian-humanimmunodeficiency virus (SHIV) replication (Amara, R. R. et al. 2002 JVirol 76:6138-6146; Ourmanov, I. et al. 2000 J Virol 74:2740-275;Polacino, P. et al. 1999 J Virol 73:618-630; Polacino, P. S. et al. 1999J Virol 73:8201-8215). However, the experiments were performed withenvelopes in the immunogens and challenge viruses that were geneticallymatched, raising questions about the practical relevance of thoseobservations. The present studies were initiated in the SHIV-rhesusmonkey model to evaluate a plasmid DNA prime-recombinantreplication-defective adenovirus (ADV) boost immunization strategy foran HIV vaccine. Further, these experiments were done to evaluate thecontribution to protection of envelope immunogens that are geneticallydisparate from the challenge virus. The findings in these studiesdemonstrate the potency of this vaccine regimen and indicate thatT-lymphocyte immunity to Env can broaden the protective cellular immuneresponse to an AIDS virus isolate independent of the sequence of the Envimmunogen.

Materials And Methods

Antibody binding and neutralization assays. HIV-1 gp120-specific bindingantibodies were quantified by enzyme-linked immunosorbent assay asdescribed previously (Crawford, J. M. et al. 1999 J Virol73:10199-10207). Immunoplates (MaxiSorb F96) (Nunc, Roskilde, Denmark)were coated with BaL-gp120 (Quality Biological, Inc., Gaithersburg,Md.), IIIB-gp120 (Advanced Biotechnologies, Inc., Columbia, Md.), orKB9-gp120 (kindly provided by Patricia Earl, National Institutes ofAllergy and Infectious Diseases, Bethesda, Md.). Antibody detection wasaccomplished with alkaline phosphate-conjugated, goat anti-monkeyimmunoglobulin G (IgG) (whole molecule; Sigma Chemical Co, St. Louis,Mo.). Neutralizing antibodies were measured in MT-2 cells as describedpreviously (Crawford, J. M. et al. 1999 J Virol 73:10199-10207).Briefly, 50 μl of cell-free SHIV-89.6P virus containing 500 50% tissueculture infective doses and grown in human peripheral blood mononuclearcells (PBMCs) was added to multiple dilutions of test plasma in 150 μlof growth medium in triplicate. These mixtures were incubated for 1 hbefore the addition of 5×10⁴ MT-2 cells. Infection led to extensivesyncytium formation and virus-induced cell killing in approximately 6days in the absence of neutralizing antibodies. Neutralizing titers werecalculated as the reciprocal dilution of plasma required to protect 50%of cells from virus-induced killing as measured by neutral red uptake.

Construction of synthetic SIV and HIV-1 genes. The synthetic SIVmac239gag-pol-nef gene was prepared by using a strategy similar to that usedto construct a previously described HIV vaccine vector (Huang, Y. et al.2001 J Virol 75:4947-4951). Briefly, the protein sequences of Gag, Pol,and Nef from SIVmac239 (GenBank accession no. M33262) were reversetranslated with the GCG package (Genetics Computer Group, Inc., Madison,Wis.) with codons typically utilized in human cells. Oligonucleotidescovering 5169 DNA by of the theoretical gene with 5′ SalI and 3′ BamHIsites and a consensus Kozak sequence were synthesized (GIBCO LifeTechnologies) from multiple fragments, each 75 by long with 25nucleotides (nt) of overlap. The codon-modified gag-pol-nef gene wasassembled by PCR with Pwo (Boehringer Mannheim) and Turbo Pfu(Stratagene) high-fidelity DNA polymerase. The PCR conditions wereoptimized with a PCR optimization kit (Stratagene) on a gradientRobocycler (Stratagene). The full-length synthetic gag-pol-nef gene wascloned into the SalI and BamHI site of the mammalian expression vector,pVR1012, and confirmed by DNA sequencing.

A synthetic 89.6P gp145ΔCFI Env gene was made analogously to a previousHIV vector (Huang, Y. et al. 2001 J Virol 75:4947-4951; Xu, L. et al.1998 Nat Med 4:37-42). Briefly, the protein sequence of the 89.6Penvelope (GenBank accession no. U89134) was reverse translated asdescribed above. Oligonucleotides covering 1,950 DNA by of thetheoretical gene, with a 5′ XbaI, a consensus Kozak sequence, and 3′BamHI site, were synthesized (GIBCO Life Technologies): each fragmentwas 60 by in length with 20 nt of overlap. In this modified envelopegene, the sequence from nt 1501 (amino acids [aa] 501, R) to 1602 (aa534, T) and nt 1771 (aa 591, M) to 1851 (aa 617, V) with respect tostart codon ATG (A as nt 1) were deleted. This deletion removes thecleavage site and fusion peptide for the envelope as well as part of theinterspace between the two heptad repeats. The protein was terminated atnt 2124 (aa 702, I). The amino acid at 617 was changed to E from D dueto the creation of XhoI cloning sites. The codon-modified gp145ΔCFI genewas assembled by PCR as described above. The synthetic gp145ΔCFI genewas cloned into the XbaI and BamHI sites of the mammalian expressionvector pVR1012, and the sequence was confirmed by DNA sequencing. Thesynthetic 89.6 Pgp140ΔCFI gene was derived from the gp145ΔCFI plasmidwith introduction of a termination codon after nt 2046 (aa 676, W).

The synthetic CCR-5-tropic clade B immunogen was derived from both HXB2and Bal strain envelopes. The protein sequence of the clade B Envglycoprotein (gp160) from HXB2 (X4-tropic; GenBank accession no. K03455)was used to create a synthetic version of the gene (X4gp160/h). Thenucleotide sequence of X4gp160/h shows little homology to the HXB2 gene,but the protein encoded is the same, with the following amino acidsubstitutions: aa 53 (phenylalanine→leucine), aa 94 (asparagine→asparticacid), aa 192 (lysine→serine), aa 215 (isoleucine→asparagine), aa 224(alanine→threonine), aa 346 (alanine→aspartic acid), and aa 470(proline→leucine). These seven amino acid substitutions were present inthe Los Alamos sequence database at the time those genes weresynthesized. To produce an R5-tropic version of the envelopeglycoprotein (R5gp160/h), the region encoding HIV-1 envelopeglycoprotein aa 205 to 361 from X4gp160/h (VRC-3300, described in WO02/32943) was replaced with the corresponding region from the BaL strainof HIV-1 (GenBank accession no. M68893, again using human preferredcodons). The full-length R5-tropic version of the envelope gene frompR5gp160/h (VRC-3000, described in WO 02/32943) was terminated after thecodon for aa 704. The truncated envelope glycoprotein (gp145) containedthe entire SU protein and a portion of the TM protein, including thefusion domain, the transmembrane domain, and regions important foroligomer formation. (H1 and H2 and their interspace are required foroligomerization.) Subsequently, the fusion and cleavage domains from aa503 to 536 were deleted. The interspace between H1 and H2 from aa 593 to620 was also deleted. The gp140 ΔCFI version was derived from thissequence by introduction of a termination codon as previously described(Chakrabarti B K et al. 2002 J Virol 76:5357-5368).

Construction and purification of the rADVs. Recombinant ADVs (rADVs)were generated by a modification of a previously published method (Ohno,T. et al. 1994 Science 265:781-784; Sullivan, N. J. et al. 2000 Nature408:605-609). Briefly, the synthetic SIVmac239 gag-pol adapted from thesequence described above (terminated at aa 1451) was cut with SalI,blunted, and then digested with BamHI, after which it was subcloned intothe blunted EcoRV and BamHI sites of the shuttle plasmid pAdAdaptCMVmcs.Synthetic HIV-1 gp140ΔCFI adapted from the sequence described above wassubcloned into the shuttle vector by using the XbaI and BamHI sites.293T cells were plated onto six-well plates and cultured to about 30%confluence, and then cotransfected with 2 μg of twice-cesiumchloride-purified and linearized shuttle plasmid with ADV cosmid by thecalcium phosphate method. After 7 to 12 days, the supernatant containingrecombinant adenovirus was collected from the cell lysate with freezingand thawing at least three times in 0.6 ml of Tris-HCl, pH 8.0. Theproduction of recombinant adenovirus was scaled up by infection of 293Tcells with the virus-containing supernatant. The viruses were purifiedby cesium chloride, aliquoted as 10¹² particles/ml, and stored inphosphate-buffered saline (PBS) with 13% glycerol at −20° C. for futureuse.

Expression of plasmid and rADV Env vaccine constructs. Expression ofplasmids encoding gp145ΔCFI(R5) and gp145ΔCFI(89.6P) was measured aftertransfection of 293T cells (in a six-well-dish) with a calcium phosphatetransfection reagent (Invitrogen) with 2 μg of each plasmid. Forty-eighthours after transfection, cells were collected, lysed in cell lysisbuffer (50 mM HEPES, 150 mM NaCl, 1% NP-40, 1× protease inhibitorcocktail [Roche]), and resolved by 4 to 15% polyacrylamide gradientsodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins weretransferred onto a nitrocellulose membrane (Bio-Rad), followed byWestern blot analysis with human HIV IgG as the primary antibody at a1:2,000 dilution. For comparison of the rADVs expressing these Envimmunogens, A549 cells were infected at 5,000 particles/cell.Forty-eight hours after infection, cell lysates were prepared andWestern blotting was performed as described above.

ELISPOT assays. Ninety-six well multiscreen plates were coated overnightwith 100 μl (per well) of 5 μg/ml anti-human gamma interferon (IFN-γ)(B27; BD Pharmingen) in endotoxin-free Dulbecco's phosphate-bufferedsaline (D-PBS). The plates were then washed three times with D-PBScontaining 0.25% TWEEN™ 20 (D-PBS/Tween), blocked for 2 h with D-PBScontaining 10% fetal bovine serum to remove the TWEEN™ 20, and incubatedwith peptide pools and 2×10⁵ PBMCs in triplicate in 100-μl reactionvolumes. Individual peptide pools covered the entire SIVmac239 Gag, Nef,and Poi proteins and both the HIV-1 HXB2/BaL and HIV-1 89.6P (KB9) Envproteins. Each pool comprised 15-aa peptides overlapping by 11 aa,except for the HIV-1 89.6P Env pool, which comprised 20-aa peptidesoverlapping by 10 aa. Each pool contained no more than 130 peptides.Each peptide in a pool was present at a concentration of 1 μg/ml.Following an 18-h incubation at 37° C., the plates were washed ninetimes with D-PBS/TWEEN™ and once with distilled water. The plates werethen incubated with 2 μg of biotinylated rabbit anti-human IFN-γ/ml(Biosource) for 2 h at room temperature, washed six times with CoulterWash (Beckman Coulter), and incubated for 2.5 h with a 1:500 dilution ofstreptavidin-alkaline phosphate (Southern Biotechnology). After fivewashes with Coulter Wash and one wash with PBS, the plates weredeveloped with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphatechromogen (Pierce), stopped by washing with tap water, air dried, andread with an enzyme-linked immunospot (ELISPOT) reader (HitechInstruments) using Image-Pro Plus image processing software (version4.1) (Media Cybernetics, Des Moines, Iowa). The number of spot-formingcells (SFC) per 10⁶ PBMCs was calculated. Medium background levels wereconsistently less than 15 SFC/10⁶ PBMCs.

CD4⁺ T-lymphocyte counts and viral RNA levels. Counts of CD4^(±) Tlymphocytes were determined by monoclonal antibody staining and flowcytometry. Plasma viral RNA levels were measured by an ultrasensitivebranched DNA (bDNA) amplification assay with a detection limit of 500copies per ml (Bayer Diagnostics).

Statistical analysis. The Kruskal-Wallis test for three or four groups(or its equivalent Wilcoxon rank sum test for two groups) was used tocompare the CD4 T-lymphocytes, peak viral RNA, set point viral RNA, andELISPOT counts between vaccine groups. The Wilcoxon test for censoreddata was used to compare time to detectable neutralizing antibodiesbetween vaccine groups. The Fisher exact test was used to compare thepresence of detectable neutralizing antibodies at day 20 or within thefirst 42 days. Linear regression (ordinary least squares) was used torelate neutralizing antibodies and ELISPOT counts to CD4 T-lymphocytecounts and (separately) to log₁₀ plasma viral RNA; the Wald test wasused to obtain significance levels. Power calculations for theKruskal-Wallis and Wilcoxon tests were based on the fact that the worstasymptotic relative efficiency of these tests versus Gaussian-basedtests is 0.86.

Results

Twenty-four Indian-origin rhesus monkeys, none of them expressing themajor histocompatibility complex class I allele Mamu-A*01, were analyzedin four experimental groups that received DNA priming followed by rADVvector boosting with the following immunogens: (i) control, (ii)Gag-Pol-Nef with no Env (mock), (iii) Gag-Pol-Nef with SHIV-89.6P Env(matched), or (iv) Gag-Pol-Nef with HXB2/BaL Env (mismatched). The DNAplasmid used in this study encoded a Gag-Pol-Nef fusion protein, butbecause of the instability of rADV constructs expressing Gag-Pol-Nef,the ADV vectors used in this study expressed only Gag-Pol. All HIV orSIV genes used in these vaccine constructs were codon modified aspreviously described to optimize expression in mammalian cells(Chakrabarti, B. K. et al. 2002 J Virol 76:5357-5368; Huang, Y. et al.2001 J Virol 75:4947-4951). A modified form of the env gene, withmutations in the cleavage site, fusion, and interhelical domains (ΔCFI),shown to increase antibody responses to Env, was used in all expressionvectors. Since these monkeys were eventually challenged with SHIV-89.6P,we refer to the HIV-1 89.6P Env immunogens as “matched” and the HIV-1HXB2/BaL Env immunogens as “mismatched.” To produce the HXB2/BaL Env,the region encoding aa 205 to 361 from the HXB2 Env was replaced withthe corresponding region from the BaL strain of HIV-1. In fact, the89.6P and HXB2/BaL ΔCFI Env proteins are only 81% identical. The ADVvector contained a deletion in E1 to render the vector replicationdefective and a partial deletion/substitution in E3 that disrupts thecoding sequences for the E3 proteins (Crawford, J. M. et al. 1999 JVirol 73:10199-10207; Ourmanov, I. et al. 2000 J Virol 74:2740-2751).The rADV expressing either the HXB2/BaL or 89.6P gp140 ΔCFI was made asdescribed previously (Polacino, P. et al. 1999 J Virol 73:618-630;Polacino, P. S. et al. 1999 J Virol 73:8201-8215). The related gag-polor identical env cDNA inserts were introduced and matched to theimmunogens in the plasmid used for DNA priming as previously described(Amara, R. R. et al. 2002 J Virol 76:6138-6146; Barouch, D. H. et al.2000 Science 290:486-492). Each plasmid DNA was deliveredintramuscularly as a 4-mg inoculum with a needleless Biojector device(Biological; Bioject Medical Technologies, Inc., Beckminister, N.J.) ona schedule of weeks 0, 4, and 8. The levels of in vitro expression ofthe HXB2/Bal and 89.6P env genes were comparable in both the plasmid andrADV vaccine constructs (FIG. 18). A single inoculation of 10¹²particles of each rADV construct was given intramuscularly to eachmonkey on week 26.

The immunogenicity of these vaccine constructs was assessed by antibodybinding, virus neutralization, and pooled-peptide ELISPOT assays. Plasmaobtained 2 weeks after the rADV boost was assessed for BaL and 89.6Pgp120 binding and for neutralization of the SHIV-89.6P challenge virus.While the Env-immunized monkeys developed high-titer antibodies to theimmunizing BaL or 89.6P gp120, plasma from week 28 of the study, thetime of peak ELISA titer antibody responses, failed to neutralize thechallenge virus SHIV-89.6P.

ELISPOT responses by the PBMCs of all monkeys receiving experimentalimmunogens were robust (FIG. 19). Cellular immunity to SIV Gag, Poi, andNef was generated in all groups of vaccinated monkeys, and that to HIV-189.6P and HXB2/BaL Env was generated in monkeys receiving theserespective Env immunogens. Monkeys injected with the mock Env (emptyvectors) did not develop Env-specific cellular immunity. Mean totalvaccine-elicited PBMC ELISPOT responses to all viral proteins 2 weeksafter the final plasmid DNA inoculations were 942±294 SFC (mean±standarderror) in the matched Env group, 1,588±554 SFC in the mismatched Envgroup, and 1,255±264 SFC in the mock Env group. Two weeks after boostingwith the rADV vectors, total ELISPOT responses were 2,892±1,116,3,993±1,000, and 3,800±984 SFC in these respective groups, a >2.5-foldincrease over the cellular immune responses elicited by DNA primingalone. These responses represented both CD4⁺ and CD8⁺ T-lymphocyteresponses, as demonstrated in ELISPOT assays performed on unfractionatedand CD8⁺ T-lymphocyte-depleted PBMCs from the monkeys (FIGS. 20A and B).While the responses declined in subsequent weeks, high-frequencyresponses were still detected in PBMCs of the monkeys at the time ofviral challenge (1,581±535, 1,908±557, and 1,092±400 SFC, respectively)in these three groups of monkeys. Thus, this vaccine regimen elicitedhigh-frequency CD4⁺ and CD8⁺ T-lymphocyte responses to multiple viralproteins. No statistically significant differences in total ELISPOTresponses were observed between the three groups of experimentallyvaccinated monkeys. The particularly high total SFC responses of thePBMCs of the monkeys in the mock Env group of animals reflectedidiosyncratically high responses to the Poi protein (FIG. 20A).Importantly, there were no significant differences between groups ofmonkeys in the magnitude of their Gag- and Pot-specific ELISPOTresponses as determined by comparison with a Mann-Whitney t test.

All monkeys were challenged intravenously with 50 50% monkey infectivedoses SHIV-89.6P on week 38, 12 weeks following the rADV boost, and weremonitored for clinical, virologic, and immunologic sequelae ofinfection. SHIV-89.6P infection causes a precipitous decline inperipheral blood CD4⁺ T lymphocytes in approximately 75% ofimmunologically naive rhesus monkeys, and selected vaccine strategiescan generate immune responses that blunt this CD4⁺ T-lymphocyte loss(Amara, R. R. et al. 2001 Science 292:69-74; Barouch, D. H. et al. 2000Science 290:486-492; Reimann, K. A. et al. 1996 J Viral 70:6922-6928;Shiver, J. W. et al. 2002 Nature 415:331-335). We therefore monitoredperipheral blood CD4⁺ T-lymphocyte counts as an indicator of theclinical status of the monkeys following SHIV-89.6P infection (FIG. 21).A profound loss of CD4⁺ T lymphocytes was observed in all controls,while substantial blunting of that CD4⁺ T-lymphocyte depletion was seenin four of the six monkeys receiving the vaccinations with SIVGag-Pol-Nef plus mock Env. Therefore, as expected based on previousstudies (Amara, R. R. et al. 2001 Science 292:69-74; Barouch, D. H. etal. 2000 Science 290:486-492; Shiver, J. W. et al. 2002 Nature415:331-335), vaccine-mediated protection against clinical sequelae ofSHIV-89.6P infection was conferred by the Gag-Pol-Nef-containingimmunogens. The two groups of vaccinated monkeys that received HIV-1 Envin addition to SIV Gag-Pol-Nef immunogens demonstrated even moreimpressive protection against CD4+ T-lymphocyte loss than the monkeysreceiving only the SIV Gag-Pol-Nef immunogens (FIG. 21). The meanperipheral blood CD4⁺ T-lymphocyte counts on day 168 postchallenge inthe groups of experimentally vaccinated monkeys were 363±100(mean±standard error) in the mock Env-vaccinated animals, 772±111 in thematched Env-vaccinated animals, and 706±76 in the mismatchedEnv-vaccinated animals, documenting that statistically significantprotection against CD4⁺ T-lymphocyte loss was afforded by inclusion ofan Env component in the vaccine (P=0.03, Kruskal-Wallis test).Importantly, the monkeys that received the mismatched Env immunogensshowed comparable protection to those injected with the matched Envimmunogens.

Viral replication in the SHIV-89.6P-challenged monkeys was assessed byquantitating viral RNA in their plasma by using a bDNA assay (FIG. 22).Since only 15% of immunologically naïve rhesus monkeys control thisvirus to undetectable levels following infection, the plasma viral RNAlevels at both peak and steady state or set point in experimentalanimals provide a measure of vaccine-mediated containment of virus. Themedians of peak viral loads in the four groups of monkeys were 1×10⁸(control), 6×10⁶ (mock Env), 4×10⁶ (matched Env), and 1×10⁶ (mismatchedEnv). Thus, the control vaccines had significantly higher peak viralloads than the vaccinated monkeys (Kruskal-Wallis test, P=0.01).However, the three groups of experimentally vaccinated monkeys did notdiffer significantly in their peak viral loads (P=0.28, Kruskal-Wallistest).

The group of monkeys that received SIV Gag-Pol-Nef plus mismatched Envimmunogens also demonstrated better containment of virus at set pointthan the monkeys receiving SIV Gag-Pol-Nef plus mock Env immunogens. Thelog copies of plasma viral RNA on day 168 postchallenge in the groups ofexperimentally vaccinated monkeys were 3.70±0.52 (mean±standard error)in the mock Env-vaccinated animals, 3.61±0.35 in the matchedEnv-vaccinated animals, and 2.38±0.18 in the mismatched Env-vaccinatedanimals, with statistically significant lower plasma viral RNA levelsafforded by inclusion of a mismatched Env component in the vaccine(P=0.04, Kruskal-Wallis test). A trend toward an association betweentotal SFC responses both pre- and postchallenge and postchallenge viralload was observed. The absence of a significant difference in plasmaviral RNA levels between the groups of experimentally vaccinated monkeysreceiving the matched Env immunogens and those receiving the mock Envimmunogens may reflect unusually low T-cell responses to the Gagimmunogens in the matched Env-vaccinated animals (FIG. 20).

To analyze the mechanism mediating improved protection against CD4⁺T-lymphocyte loss in the Env-immunized monkeys, the antiviral humoralimmune response was evaluated. Anti-Env antibody could potentiallycontribute to protection by neutralizing infectious virus at the time ofchallenge. Alternatively, a rapidly evolving anamnestic neutralizingantibody response after infection could contribute to the control ofviral spread. None of the vaccinated monkeys had detectable plasmaneutralizing antibodies at the time of challenge, indicating thatvaccine-elicited preexisting neutralizing antibody did not contribute toviral containment. The evolution of an antibody response thatneutralized the challenge virus SHIV-89.6P was monitored on a weeklybasis in vaccinated monkeys after viral challenge (FIG. 23). At 3 weekspostchallenge, three animals in the matched Env group, but none in themock or mismatched Env groups, showed an anamnestic response to thechallenge virus. However, there was no statistically significantdifference between the three groups of experimentally vaccinated monkeysin time to the detection of neutralizing antibody or number of animalsdeveloping detectable neutralizing antibody responses. Of note, thestatistical tests applied to these data have very little power to detectdifferences among groups because of the small number of monkeys in eachexperimental group. Thus, it remains possible that neutralizingantibodies had an effect that we were unable to detect.

To evaluate further whether the emergence of a neutralizing antibodyresponse was associated with either clinical or virologic eventsfollowing SHIV-89.6P challenge, a linear regression analysis wasperformed to evaluate the association of detectable neutralizingantibodies with either plasma viral RNA levels or peripheral blood CD4⁺T-lymphocyte counts. In fact, these variables showed no significantassociation with the development of neutralizing antibody, whetherassessed on the basis of its emergence over time or its detection at asingle time during the first 6 weeks following challenge. Therefore, wewere unable to demonstrate that neutralizing antibodies directed againstSHIV-89.6P contributed to viral containment after challenge. Finally,the fact that the mismatched Env group appeared to control plasmaviremia more effectively than the matched Env group further suggeststhat neutralizing antibodies did not substantially contribute to viralcontainment.

To examine the possible contribution of Env-specific T-cell responses toprotective immunity in these monkeys, PBMC cellular immune responsesfrom the four groups of experimental monkeys were assessed 1 weekfollowing rADV boosting for cellular immunity to a pool of HIV-1 89.6PEnv peptides in an ELISPOT assay (FIG. 24, top panel). The meanresponses were 449±122 SFC (mean±standard error) in the matched Envgroup, 730±306 SFC in the mismatched Env group, and 13±8 SFC in the mockEnv group. The apparent higher PBMC SFC response in the HXB2/BalEnv-vaccinated monkeys to 89.6P Env than to HXB2/BaL Env does notachieve statistical significance. Thus, impressive cellular immunity wasseen in PBMCs of the HXB2/Bal Env-immunized monkeys that reacted withthe Env of the challenge virus.

Since cellular immune responses that develop following initial infectioncontribute to containment of AIDS virus spread (Barouch, D. H. et al.2000 Science 290:486-492), we reasoned that differences in theseresponses to Env between the groups of vaccinated monkeys may explainthe differences in their clinical outcomes. Therefore, we assessed HIV-189.6P Env-specific T-cell responses in these monkeys 3 and 10 weeksfollowing SHIV-89.6P challenge (FIG. 24). Strikingly, PBMCs of themonkeys that received either matched or mismatched Env immunogensdeveloped dramatically higher ELISPOT responses to these Env peptidesthan did the monkeys that received the mock Env immunizations (P=0.002,Wilcoxon rank sum test). Therefore, a strong association was seenbetween the generation of Env-specific T-cell responses postchallengeand the inclusion of either matched or mismatched Env immunogens in thevaccine regimens of these monkeys.

Discussion

This study demonstrates that HIV Env contributes to immune protection ina simian lentivirus challenge model. Importantly, protection wasobserved when the Env immunogen was matched or mismatched relative tothe challenge viral strain. These findings indicate that it is advisableto include this gene product in vaccine candidates, even if suchvaccines do not elicit a broadly neutralizing antibody response.

The present study provides a strong rationale for including Env antigensin HIV vaccines that advance into human efficacy trials. The currentfocus of effort on Env immunogen design continues to center onmodifications that will induce broadly neutralizing antibodies (Mascola,J. R. 2003 Curr Mol Med 3:209-216). Such modifications will no doubtfurther enhance vaccine efficacy if successful. However, this studyindicates that the inclusion of Env as a vaccine immunogen, even if itdoes not induce a broadly neutralizing antibody response, contributes tovirus containment and immune preservation. The enhanced breadth ofcellular immunity appears sufficient to improve the clinical protectionconferred by vaccination.

Part V Multiclade HIV-1 Envelope Immunogens Elicit Broad Cellular andHumoral Immunity in Rhesus Monkeys

Abstract

The development of an HIV-1 vaccine that elicits potent cellular andhumoral immune responses that recognize divergent strains of HIV-1 willbe critical for combating the global AIDS epidemic. The present studieswere initiated to examine the magnitude and breadth of envelope(Env)-specific T lymphocyte and antibody responses generated by vaccinescontaining either a single or multiple genetically distant HIV-1 Envimmunogens. Rhesus monkeys were immunized with DNA prime/rAd boostvaccines encoding a Gag/Pol/Nef polyprotein in combination with either asingle Env or with a mixture of clade A, clade B, and clade C Envs.Monkeys receiving the multiclade Env immunization developed robustimmune responses to all vaccine antigens, and importantly, a greaterbreadth of Env recognition than monkeys immunized with vaccinesincluding a single Env immunogen. All groups of vaccinated monkeysdemonstrated equivalent immune protection following challenge with thepathogenic simian human immunodeficiency virus (SHIV)-89.6P. These dataindicate that a multicomponent vaccine encoding Env proteins frommultiple clades of HIV-1 can generate broad Env-specific T lymphocyteand antibody responses without antigenic interference. This studydemonstrates generating protective immune responses by vaccination withgenetically diverse isolates of HIV-1.

Introduction

The extreme genetic diversity of human immunodeficiency virus type 1(HIV-1) envelope (Env) poses a daunting challenge for the creation of aneffective AIDS vaccine (Letvin, N. L et al. 2002 Annu Rev Immunol20:73-99). While Env is the principal target for HIV-1-specific antibodyresponses, it also serves as a potent T cell immunogen (See PART IV). Anideal HIV-1 vaccine should elicit potent cellular and humoral immunitycapable of recognizing a diversity of viral isolates (Mascola, J. R.,and G. J. Nabel. 2001 Curr Opin Immunol 13:489-95; Nabel, G. J. 2001Nature 410:1002-7). However, the extraordinary genetic variation ofHIV-1 Env worldwide may make it impossible to create an effectivevaccine using only a single Env gene product.

While many of the promising AIDS vaccine candidates currently underinvestigation in nonhuman primates and early phase human clinical trialsutilize Env immunogens derived from a single HIV-1 primary isolate(Graham, B. S. 2002 Annu Rev Med 53:207-21), this approach hassignificant limitations. Although these vaccines generate potentcellular and humoral immune responses against HIV-1 Env, it is likelythat the breadth of immunity elicited by a single Env immunogen will noteffectively confer protection against divergent strains of HIV-1. It is,however, not feasible to undertake the development of multiple country-or Glade-specific vaccines. Moreover, such region-specific vaccineswould likely not protect against unrelated strains that might be newlyintroduced into a population.

One strategy for creating a single HIV-1 vaccine for worldwide use is toemploy representative immunogens from multiple clades of HIV-1 in asingle vaccine formulation (Nabel, G. J. et al. 2002 Science 296:2335).Such a multiclade vaccine would contain Env immunogens relevant to themajority of HIV-1 infections worldwide and could be feasibly tested.However, it is not clear whether a multicomponent vaccine encodingantigens from various clades of HIV-1 would elicit antiviral immunitygreater than or equal to a vaccine employing a single Env immunogen, andwhether a complex mixture of immunogens would result inantigenic-interference and diminished immune protection (Kjerrstrom, A.et al. 2001 Virology 284:46-61).

The present studies utilized the simian human immunodeficiency virus(SHIV)/rhesus monkey model to investigate the breadth and magnitude ofimmunity elicited by a DNA prime/recombinant adenovirus boost vaccinecontaining Gag/Pol/Nef and either single clade or multiple clade Envimmunogens. Our findings demonstrate that a multiclade Env vaccineelicits potent cellular and humoral immune responses with greaterbreadth than can be generated with immunizations performed with a singleEnv immunogen.

Materials and Methods

Immunizations and challenge of rhesus monkeys. Thirty adultIndian-origin rhesus monkeys (Macaca mulatta) were maintained in afacility accredited by the Association for the Assessment andAccreditation of Laboratory Animal Care in accordance with theguidelines of the Institutional Animal Care and Use Committee forHarvard Medical School and the Guide for the Care and Use of LaboratoryAnimals. Monkeys were divided into five groups of six animals. Eachexperimental group included two monkeys expressing the MHC class Iallele Mamu-A*01.

Plasmid DNA and recombinant adenovirus (rAd) vaccine vectors wereconstructed as previously described (see PARTS II and IV), andadministered by intramuscular injection using a needle-free Biojectorsystem and a no. 3 syringe (Bioject, Portland, Oreg.) as outlined inTable 3. Each plasmid DNA or rAd vaccine vector was split into twoaliquots of 0.5 ml each, and delivered into each quadriceps muscle.Control monkeys were similarly immunized with sham DNA and sham rAdvectors. At week 42, all monkeys received an intravenous challenge with50 50% monkey infective doses (MID₅₀) of SHIV-89.6P.

IFN-γ ELISPOT assays. IFN-γ ELISPOT assays were performed as describedabove in PART IV. Freshly isolated PBL were plated in triplicate at2×10⁵ cells/well in 100 μl final volume with either medium alone orpeptide pools. Peptide pools covered the entire SIVmac239 Gag, Nef, andPol proteins, and the HIV-1 clade A, clade B, clade C, and 89.6P Envproteins. Each pool was comprised of 15 amino acid peptides overlappingby 11 amino acids, but for the HIV-1 89.6P Env pool, which was comprisedof 20 amino acid peptides overlapping by 10 amino acids. Pol and Envpeptides were each split into two separate pools such that each poolcontained no more than 130 peptides. Each peptide in a pool was presentat a concentration of 1 μg/ml. The mean number of spots from triplicatewells was calculated for each animal and adjusted to represent the meannumber of spots per 10⁶ PBL. Data are presented as the mean number ofantigen-specific spots per 10⁶ PBL from 6 monkeys per group.

HIV-1 Envelope Antibody ELISA. Vaccine Research Center (VRC) plasmids5304, 2801, and 5308 (which encode HIV-1 gp145 clade A, clade B, andclade C Env, respectively) (described in WO 02/32943) were expressed in293 cells and purified for the major protein product. Optimizedconcentrations of the recombinant antigens (37.5 ng/well) were coatedonto Immunol-2 HB microtiter plates (Thermo Labsystems, Milford, Mass.)overnight at 4° C. Serial dilutions of monkey plasma were done induplicate wells and incubated for 2 hours at 37° C. Biotin labeledanti-monkey IgG/IgA/IgM (Rockland Immunochemicals, Gilbertsville, Pa.)was added for 1 hr at 37° C. Streptavidin-HRPO (KPL, Gaitherburg, Md.)was added to wells for 30 minutes at room temperature, followed by TMBsubstrate (KPL) for 30 minutes at room temperature. Endpoint titers foreach animal were established as the last dilution with apre-immunization corrected OD>0.2. Data are presented as the geometricmean titer from 6 monkeys per group+/−SEM.

Virus isolates and neutralization assays. A total of 30 HIV-1 isolateswere studied: 11 clade B, 11 clade C and 8 clade A. Viruses wereobtained from the NIH AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, NIH, except as specifically noted below. Allclade B viruses were primary isolates except the T-cell line adaptedHxB2, which is the molecular clone of HIV-IIIB. BRO7 was provided byDana Gabuzda of the Dana-Farber Cancer Institute. It is a chimericinfectious molecular clone of NL4-3 that contains a near full-length Envgene that was cloned directly from brain tissue of an AIDS patient(Ohagen, A. et al. 2003 J Virol 77:12336-45). Clade B primary isolate6101, previously called P15 (Bures, R. et al. 2000 AIDS Res HumRetroviruses 16:2019-35) and clade C viruses DU123, DU151, S007 and 5080were provided by David Montefiori (Duke University Medical Center). Theclade C viruses were obtained from HIV-1 infected patients in SouthAfrica (Du prefix) or Malawi (S prefix) and have been previouslydescribed (Bures, R. et al. 2002 J Virol 76:2233-44). TV1 (clade C) wasprovided by David Montefiori and Estrelita Janse Van Rensburg(University of Stellenbosch, South Africa). GS14 is an infectiousmolecular clone of an Ethiopian clade C virus that was provide byFrancine McCutchan and colleagues from the US Military HIV Researchprogram. Clade A viruses DJ263 and 44951 were primary virus isolatesprovided by researchers from the US Military HIV Research program. TheUG29 isolate had been previously passaged into H9 cells, and wouldtherefore be considered a T-cell line adapted virus.

Virus neutralization assays were performed using a single round ofinfection flow cytometric assay using previously described methods(Mascola, J. R. et al. 2002 J Virol 76:4810-21). This assay detectsHIV-1 infected T-cells by intracellular staining for HIV-1 p24 Gagantigen (p24-Ag). A protease inhibitor is used to prevent secondaryrounds of virus replication. The percent virus neutralization mediatedby each immune plasma was derived by calculating the reduction in thenumber of p24-Ag positive cells in the test wells with immune sera,compared to the number of p24-Ag positive cells in wells containingpre-immune plasma from the corresponding animal. Plasma from the sixsham immunized monkeys was included for analysis, and these data areshown in the results section. All plasma samples were also testedagainst an amphotropic murine leukemia virus (MuLV) to test fornon-HIV-1 specific plasma effects. The MuLV reporter viruses encodedgreen fluorescent protein (GFP) and infected T-cell cells were detectedby expression of GFP rather than expression of p24-Ag (Mascola, J. R. etal. 2002 J Virol 76:4810-21).

Quantitation of plasma viral RNA levels and CD4⁺ T lymphocyte counts.Plasma viral RNA levels were measured by an ultrasensitive branched DNAamplification assay with a lower detection limit of 125 copies per ml(Bayer Diagnostics, Berkeley, Calif.). Peak plasma viral load wasmeasured on day 16 post-SHIV-89.6P challenge in all vaccinated andcontrol monkeys. Set point plasma viral RNA levels were calculated asthe median of values measured at six time points between days 85 and 169post-challenge. The percentage of CD4⁺ T lymphocytes in the peripheralblood of infected monkeys was deteimined by monoclonal antibody stainingand flow cytometric analysis. Briefly, freshly isolated PBL were stainedwith anti-CD3 APC (FN18), anti-CD4 PE (19Thy5D7), and anti-CD8 FITC(SK1, BD Biosciences, Mountain View, Calif.). Samples were acquiredusing a FACSCalibur flow cytometer and data analyzed using CellQuestsoftware (BD Biosciences).

Statistical Analysis. The nonparametric Wilcoxon rank sum test was usedto compare CD4⁺ T lymphocytes, peak viral RNA, and set point viral RNAbetween monkeys in the non-vaccinated and vaccinated groups. All testswere two-sided.

Results

Study design. Thirty adult rhesus monkeys were divided into fiveexperimental groups of six animals (Table 3). Groups 1-4 received threepriming immunizations at weeks 0, 4, and 8 with 4.5 mg plasmid DNAvectors expressing an SIVmac239 Gag-Pol-Nef fusion protein and plasmidDNA vectors expressing various HIV-1 Env proteins. Groups 1-3 wereimmunized with single HIV-1 Env immunogens as follows: 1) 4.5 mg clade BEnv (high clade B), 2) 1.5 mg clade B Env (low clade B), and 3) 4.5 mgclade C Env (high clade C). Group 4 monkeys were immunized with acombination of HIV-1 Env immunogens, 1.5 mg each of a clade A Env, cladeB Env, and clade C Env (clade A+B+C). At week 26, monkeys received asingle rAd boost immunization (2.0×10¹² total particles) with vectorsexpressing SIVmac239 Gag-Pol and various HIV-1 Env genes consistent withthose delivered during the DNA priming (Table 3). Groups 1-3received: 1) 1.0×10¹² particles clade B Env (high clade B), 2) 3.3×10¹¹particles clade B Env (low clade B), 3) 1.0×10¹² particles clade C Env(high clade C). Group 4 received 3.3×10¹¹ particles each of clade A,clade B and clade C Env (clade A+B+C). Group 5 monkeys were immunizedwith sham DNA and sham rAd vectors. DNA prime and rAd boostimmunizations were delivered by intramuscular injection. All plasmid DNAand rAd vectors expressed codon-modified SIVmac239 and HIV-1 genes forenhanced expression in mammalian cells. All env genes used in thesevectors were ΔCFI constructs, containing mutations in the cleavage,fusion, and interhelical domains that have previously been shown toenhance expression and immunogenicity (Chakrabarti, B. K. et al. 2002 JViral 76:5357-68). The percent amino acid identity among the HIV-1 Envimmunogens ranged from 71-76%, with the clade B and clade C Envsdemonstrating the greatest divergence.

TABLE 3 Experimental groups and immunization schedule Sham SIVGag-Pol-Nef HIV-1 Env Plasmid Group Plasmid (mg) Plasmid (mg) (mg) 1)High Clade B Env 4.5 4.5 Clade B — 2) Low Clade B Env 4.5 1.5 Clade B3.0 3) High Clade C Env 4.5 4.5 Clade C — 4) Clade A + B + C Env 4.5 1.5Clade A 1.5 Clade B 1.5 Clade C 5) Control — — 9.0 SIV Gag-Pol rAd HIV-1Env rAd Sham rAd Group (particles) (particles) (particles) 1) High CladeB Env 1.0 × 10¹² 1.0 × 10¹² Clade B — 2) Low Clade B Env 1.0 × 10¹² 3.3× 10¹¹ Clade B 6.6 × 10¹¹ 3) High Clade C Env 1.0 × 10¹² 1.0 × 10¹²Clade C — 4) Clade A + B + C Env 1.0 × 10¹² 3.3 × 10¹¹ Clade A 3.3 ×10¹¹ Clade B 3.3 × 10¹¹ Clade C 5) Control — — 2.0 × 10¹²

Cellular immune responses elicited by immunization. The cellular immuneresponses to SIV Gag and Pol and HIV-1 Envs in immunized monkeys wereassessed by pooled peptide IFN-γ ELISPOT assays using freshly isolatedPBL. Moreover, the extent of cross-clade reactivity of vaccine-elicitedEnv-specific cellular immune responses was determined by measuring PBLIFN-γ ELISPOT responses to clade A, clade B, and clade C Env peptidepools. Because these monkeys were to be challenged with SHIV-89.6P, wealso evaluated T cell recognition of a peptide pool representing theclade B 89.6P Env. Monkeys receiving the high and low dose clade B Envplasmid DNA immunogen generated cellular immune responses to all Envpeptide pools tested (FIG. 25A). The responses to both the clade B and89.6P (heterologous clade B) Env peptide pools were of a higherfrequency than those observed against the clade A or clade C Env pools.Monkeys receiving the high dose clade C Env immunogen also developedcellular immune responses to all Env peptide pools tested, but withclade C Env responses higher than those to clade A, clade B, or 89.6PEnvs. Importantly, comparable cellular immune responses to clade A, B, Cand 89.6P HIV-1 Env peptide pools were observed in PBL of the monkeysreceiving the multiclade plasmid DNA immunogens.

The DNA primed cellular immune responses of all vaccinated monkeys weredramatically augmented following the boost immunization with the rAdvaccines (FIG. 25B). While responses to all Env peptide pools wereobserved in the monkeys receiving the high dose clade B Env rAd boostimmunization, SFC responses to clade B and 89.6P peptides were higherthan those to clade A or C peptides (p=0.06 and 0.04, respectively,Wilcoxon rank sum test). The Env-specific cellular immune responses ofthe low dose clade B Env-immunized monkeys were comparable to those ofmonkeys receiving the high dose clade B Env immunogens. Thus, loweringthe dose of the Env plasmid and rAd vaccines by two thirds did notresult in major reductions in immunogenicity. The animals boosted withthe high dose clade C Env rAd construct also showed an increase in Tcell reactivity to all Env peptide series, but responses to clade Cpeptides were significantly higher than those to clade A or B peptides(p=0.04 for both). In contrast, multiclade Env immunized monkeysexhibited no bias in Env-specific cellular immune responses. Followingthe boost immunization with the clade A, clade B, and clade C Env rAdconstructs, the monkeys developed responses to the clade A, clade B,clade C, and 89.6P Env peptide pools that were of comparable magnitude(FIG. 25B). Furthermore, the magnitudes of each individualclade-specific ELISPOT response in these monkeys were comparable to theoptimal clade-specific response elicited in monkeys receiving a singleclade Env immunogen. Finally, the vaccine-elicited Env-specific T cellresponses in all groups of monkeys were durable, persisting at a highfrequency up to the time of viral challenge (FIG. 25C).

Cellular immune responses to SIV Gag and Pol were observed in allvaccinated monkeys following the DNA priming immunizations as well asfollowing the rAd boost immunizations (FIG. 26). Importantly, PBL ofmonkeys receiving the multiclade Env immunizations developed ELISPOTresponses to these SIV proteins that were comparable in magnitude tothose observed from monkeys receiving single clade Env immunogens. Thus,immunizing monkeys with the complex pool of SIV Gag-Pol and multicladeHIV-1 Env immunogens elicited cellular immune responses to all thevaccine components without evidence of antigenic interference.

Antibody responses elicited by immunization. The magnitude and breadthof humoral immune responses elicited by single clade and multiclade Envimmunizations were investigated in these monkeys following rAdadministration. Plasma samples were tested for antibody binding activityto the clade A, clade B, and clade C gp145 Env proteins by ELISA.Monkeys receiving the high dose clade B Env immunogens generatedantibody responses that bound all three Env proteins (FIG. 27); however,the highest antibody titers were against the clade B Env protein(p=0.002 and 0.13 versus clade A and C Env proteins, respectively,Wilcoxon rank sum test). A similar pattern of antibody reactivity wasobserved in the monkeys receiving the low dose clade B Envimmunizations, and lowering the dose of Env immunogen by two thirds didnot result in a substantial reduction in immunogenicity. Monkeysreceiving the high dose clade C Env immunogens similarly developedantibody responses that recognized all three Env antigens, but titersagainst the clade C Env protein were significantly higher than thoseagainst clade A or B Env proteins (p=0.004 and 0.002, respectively). Incontrast, monkeys immunized with the mixture of clade A, clade B, andclade C Env immunogens demonstrated comparable antibody responses to allthree Env proteins.

Plasma samples obtained following the rAd boost immunizations were alsotested for neutralizing activity against panels of 30 clade A, clade B,and clade C HIV-1 isolates (FIG. 28). While plasmas from all groups ofvaccinated monkeys demonstrated modest levels of neutralization againstsome HIV-1 isolates, the antibodies of monkeys immunized with a singleclade Env immunogen exhibited the highest neutralizing activity againstviruses of the same clade. Thus, plasma from the clade B immunizedanimals displayed little activity against clade A or C viruses andplasmas from clade C immunized animals did not neutralize clade Bviruses. However, there was some cross-neutralization of clade A virusesby the clade C vaccine plasma. Importantly, the multiclade Env immunizedmonkeys developed antibodies with neutralizing activity against someHIV-1 strains from all three clades, and there was no decrement in thepotency of neutralization compared to single Env immunization. Twocontrols were performed to demonstrate that the modest levels of virusneutralization observed were due to HIV-1 specific antibodies. The meanneutralization activity of plasma obtained from sham vaccinated monkeyswas consistently less than twenty percent (FIG. 28, dashed line). Inaddition, the mean activity of plasma from each of the vaccine groupsagainst a MuLV Env pseudovirus was less than 20% (shown in FIG. 28A).These data indicate that the multiclade Env immunization regimenelicited humoral immune responses of increased breadth when compared toresponses elicited by immunization with a single Env, and withoutevidence of antigenic interference.

Protection against SHIV-89.6P challenge. All monkeys received anintravenous challenge with 50 MID₅₀ SHIV-89.6P on week 42, 16 weeksfollowing the rAd boost immunization. At two weeks after viralchallenge, robust cellular immune responses to HIV-1 Env and SIV Gag andPol were detected in all groups of experimentally vaccinated monkeys butnot in control monkeys (FIG. 29). Peak plasma viral RNA levels wereobserved in all monkeys on day 16 following challenge, with median log₁₀values of 7.54 (control), 5.66 (high clade B Env), 6.83 (low clade BEnv), 6.20 (high clade C Env), and 6.46 (clade A+B+C Env) copies ofviral RNA detected (FIG. 30A). Thus, a significant reduction in the peakviral load following SHIV-89.6P challenge was observed in all groups ofexperimentally vaccinated monkeys when compared with non-vaccinatedcontrol monkeys (p values ranging from 0.002 to 0.004, Wilcoxon rank sumtest). However, no significant differences were observed in peak viralRNA levels when monkeys receiving the single clade Env or multiclade Envimmunizations were compared.

All groups of experimentally vaccinated monkeys also demonstrated betterlong term containment of virus than control monkeys, with median log₁₀set point viral RNA levels of 4.77 (control), 2.30 (high dose clade BEnv), 2.63 (low dose clade B Env), 2.28 (high dose clade C Env), and2.69 (clade A+B+C Env) copies of viral RNA measured between days 85 and169 post-challenge (FIG. 30B). No significant differences in set pointviral loads were observed among the groups of monkeys vaccinated withthe single or multiclade Env immunogens.

Peripheral blood CD4⁺ T lymphocyte counts were also measured in theinfected monkeys as a means of evaluating vaccine-mediated protectionagainst SHIV-89.6P-induced disease. Sham vaccinated control monkeysdeveloped a rapid and persistent decline in CD4⁺ T lymphocytes withinthe first 21 days following challenge (FIG. 31). All groups ofexperimentally vaccinated monkeys exhibited significant blunting of CD4⁺T lymphocyte loss between days 85 and 169 post-challenge when comparedwith control monkeys (p values ranging from 0.015 to 0.026). While therewere no significant differences in CD4⁺ T lymphocyte numbers between thegroups of vaccinated monkeys during the acute and chronic phases ofinfection, monkeys in the high dose clade B Env and multiclade Envvaccine groups demonstrated the best preservation of this lymphocytepopulation.

Discussion

A global HIV-1 vaccine must elicit effective immune responses to diverseviral isolates. In fact, broadly cross-reactive HIV-1-specific T cellimmune responses have been described. HIV-1-infected individuals developT lymphocyte responses that recognize viral sequences from a diversityof HIV-1 clades (Cao, H. et al 1997 J Viral 71:8615-23). Cross-cladereactive CTL have also been detected in uninfected volunteers who havebeen vaccinated with recombinant canarypox constructs (Ferrari, G. etal. 2000 AIDS Res Hum Retroviruses 16:1433-43). However, because thesestudies employed CTL clones or in vitro cultured PBL to assesscross-clade T cell reactivity, the true breadth of these HIV-1-specificimmune responses is unknown. In the present study we demonstrate thatimmunization of rhesus monkeys with a DNA prime/rAd boost vaccine thatincludes multiple Env immunogens elicits cellular and humoral immuneresponses that exhibit a greater breadth of Env-specific recognitionthan those observed in monkeys immunized with single Env immunogens.

PBL from monkeys immunized with single HIV-1 Env immunogens demonstratedhigh frequency cellular immune responses to peptide pools matching thevaccine-encoded Env immunogen, with lower frequency responses topeptides of Env proteins not included in the vaccine. Thesecross-reactive responses may reflect T lymphocyte recognition ofconserved viral epitopes, as well as cross-reactive recognition ofvariant epitopes that may differ by limited numbers of amino acids(Charini, W. A. et al. 2001 J Immunol 167:4996-5003; Keating, S. M. etal. 2002 AIDS Res Hum Retroviruses 18:1067-79). The highest degree ofheterologous Env recognition in this study was the reactivity of PBL ofmonkeys immunized with the clade B HXBc2/BaL Env immunogen againstpeptide pools representing 89.6P Env, a heterologous clade B Env (FIG.25). HXBc2/BaL Env shares 81% amino acid identity with 89.6P Env, andonly 75% and 72% identity, respectively, with the clade A and C Envsequences used in these immunizations. These data indicate thatimmunizing with single Env immunogens may elicit the highest frequencycross-reactive T cell responses against Envs of viruses of the sameclade.

A concern with a vaccine that includes viral proteins from multipleclades of HIV-1 is that interference between these diverse antigens maydiminish immune responses. In fact, such antigenic interference has beenobserved in vaccines that include proteins of multiple pathogens(Fattom, A et al. 1999 Vaccine 17:126-33; Insel, R. A. 1995 Ann NY AcadSci 754:35-47). Moreover, studies have shown that complex mixtures ofplasmid DNA vaccines can lead to decreased protein expression andimmunogenicity in vivo (Kjerrstrom, A. et al. 2001 Virology 284:46-61;Sedegah, M. et al. 2004 Gene Ther 11:448-56). The findings in thepresent study, however, demonstrate that including Env immunogens frommultiple clades of HIV-1 in a single vaccine can increase the breadth ofvaccine-elicited Env-specific T cell and antibody responses. Thus,monkeys immunized with the multiclade Env vaccine developed highfrequency cellular immune responses and high titer antibody responses toall vaccine-encoded Env antigens. The magnitudes of T lymphocyteresponses to the clade B and clade C Env peptide pools following the DNAprime and rAd boost with the multiclade Env immunogens were similar tothose observed in monkeys receiving the high dose single clade B or CEnv vaccines. Furthermore, no deleterious effects on the magnitude ofGag- or Pol-specific cellular immune responses were detected in themulticlade Env immunized monkeys. These results support studies in micedemonstrating that multiclade HIV-1 vaccines can elicit robust cellularand humoral immune responses to all vaccine-encoded antigens withoutevidence of antigenic-interference (PARTS I and II).

It is encouraging to note that the inclusion of clade A, B, C Envimmunogens elicits neutralizing antibodies to some clade strains notincluded in the vaccine. The present data further show that multicladeEnv immunization does not diminish vaccine-elicited immune protectionagainst SHIV-89.6P infection. Monkeys receiving DNA prime/rAd boostvaccines encoding either a single Env or multiple clade Env immunogensdemonstrated equivalent viral containment during acute and chronicinfection, and comparable preservation of CD4⁺ T lymphocytes. We havedemonstrated above (see PART V) that DNA prime/rAd boostvaccine-elicited protection against SHIV-89.6P infection is associatedwith an anamnestic antigen-specific cellular rather than neutralizingantibody responses. It therefore is not surprising that no significantdifferences in clinical protection were evident between the variousgroups of vaccinated monkeys, as they all demonstrated robustpre-challenge cellular immune responses to SIV Gag and Pol, as well assome degree of cellular immune cross-reactivity to 89.6P Env. In fact,the ELISPOT responses to 89.6P Env increased rapidly in PBL of allgroups of Env-vaccinated monkeys following challenge, suggesting thatvaccine-elicited T lymphocytes capable of recognizing 89.6P Env epitopesexpanded in response to the replicating virus (FIG. 29).

The present study demonstrates that the inclusion of viral proteins frommultiple clades of HIV-1 is a viable approach for a global HIV-1vaccine.

Part VI VRC-HIVDNA-009-00-VP

Introduction

VRC-HIVDNA-009-00-VP is a vaccine composed of four DNA plasmids encodingproteins from HIV-1 and is intended to prevent HIV-1 infection. Thevaccine has been designed to elicit immune responses against severalproteins from three HIV-1 clades. Plasmid VRC-4306 (SEQ ID NO: 20) isdesigned to express HIV-1 polyproteins (structural core protein Gag,viral polymerase Pol, and accessory protein Nef) from clade B and is 50%(by weight) of the vaccine. Plasmids VRC-5305 (SEQ ID NO: 21), VRC-2805(SEQ ID NO: 22), and VRC-5309 (SEQ ID NO: 23) express the HIV-1 Envelope(Env) glycoproteins from clade A, clade B, and clade C, respectively,and are each 16.67% (by weight) of the vaccine.

The DNA plasmid expressing HIV-1 Gag-Pol-Nef polyprotein has beenmodified to reduce toxicity through the incorporation of deletions intothe regions affecting the protease, reverse transcriptase, and integraseactivities. The amino acid sequence of the Nef protein was not modified.The amino acid sequence of the Nef protein was fused in frame from theinitiator methionine to the poi gene of HIV and is non-functional. Thesequences used to create the DNA plasmids encoding Env are derived fromthree HIV-1 CCR5-tropic strains of virus. Expression of the geneproducts is controlled by the constitutive cytomegalovirus (CMV)promoter. These DNA plasmids have been produced in bacterial cellcultures under kanamycin selection. In all cases, bacterial cell growthis dependent upon expression of the kanamycin resistance protein encodedin the plasmid DNA. Following growth of bacterial cells harboring theplasmid, the plasmid DNA is purified from cellular components. The cladeB gag-pol-nef plasmid (VRC-4306) is 9790 nucleotide pairs in length andhas an approximate molecular weight of 6.5 MDa; the clade A, B, and Cenv plasmids (VRC-5305, VRC-2805, and VRC-5309) are 6836, 6869, and 6829nucleotides in length, respectively, and have an approximate molecularweight of 4.5 MDa.

Description of the Drug Substance

1. Name of the Drug Substance: VRC-2805 (SEQ ID NO: 22) Description: Envglycoprotein, clade B Molecular Weight: 4.5 MDa Nucleotide Base Pairs:6869 2. Name of Drug Substance: VRC-4306 (SEQ ID NO: 20) Description:Gag-Pol-Nef, clade B Molecular Weight: 6.5 MDa Nucleotide Base Pairs:9790 3. Name of Drug Substance VRC-5305 (SEQ ID NO: 21) Description: Envglycoprotein, clade A Molecular Weight: 4.5 MDa Nucleotide base pairs6836 4. Name of Drug Substance VRC-5309 (SEQ ID NO: 23) Description: Envglycoprotein, clade C Molecular Weight: 4.5 MDa Nucleotide base pairs:6829

A. Production of the Gag-Pol-NefDNA Plasmids VRC-4306 [pVR1012 Gag-B(ΔFS) Pol (ΔPR ΔRT ΔIN) Nef/h]

To construct DNA plasmid VRC-4306, diagrammed in FIG. 32, the proteinsequences of the Gag, Pol, and Nef proteins from an HIV-1 clade B wereused to create a synthetic polyprotein version of the gag-pol-nef genesusing codons optimized for expression in human cells. The synthetic gaggene is from HIV-1 clade B strain HXB2 (GenBank accession number K03455,amino acids 1-432), the synthetic pol gene (pol/h) is from HIV-1 clade BNL4-3 (GenBank accession number M19921), and the synthetic nef gene(nef/h) is from HIV-1 clade B strain PV22 (GenBank accession numberK02083). The nucleotide sequence of the synthetic gag-pol-nef/h geneshows little homology to the HIV-1 gene, but the protein encoded is thesame.

In addition, mutations created in the regions that affect protease(R113G), reverse transcriptase (D341H), and integrase (D779A) reduce thepotential for functional activity. Note that GenBank accession numberM19921 has a G at position 113, but mutagenesis studies of pol geneshave shown an G at this position shows no functional activity (Loeb, D.D. et al. 1989 Nature 340:397-400). The first two amino acids of Polwere deleted in order to make the gag-pol-nef fusion gene. Nomodifications were made to the gag and nef genes.

Plasmid VRC-4306 was constructed by fusion of nef/h to the 3′ terminalof the gag-pol plasmid (VRC-4302), which is described in the WO02/32943. There were no losses or additions of amino acids created bythe fusion between pol and nef. The ATG of nef was preserved. Theconstruct was then inserted into the pVR1012 backbone using SalI andBbvCI restriction sites. The SalI (5 nt upstream from ATG) to BbvCI(4917 nt downstream from ATG) fragment contains the 5′ end and the ATGwas cloned into the SalI to BbvCI sites of the pVR1012 backbone.

A summary of the predicted VRC-4306 domains is provided in Table 4. Theplasmid is 9790 nucleotide pairs (np) in length and has an approximatemolecular weight of 6.5 MDa. The kanamycin gene is incorporated into thebacterial vector backbone as a selectable marker. The sequence ofVRC-4306 is provided as SEQ ID NO: 20.

TABLE 4 Summary of Predicted Domains of VRC-4306 Clade B gag (Δfs) polΔPR, RT, INT Nef/h Fragment Size Predicted Fragment Fragment Name orProtein Domain (bp) Location pUC derived 247  1-247 CMV-IEenhancer/promoter 638 248-885 CMV-IE 5′ UT region 244  886-1129 CMV IEintron 711 1130-1840 Synthetic linker 39 1841-1879 Gag (Δ fs) Pol (Δ PR,RT, INT) 4920 1880-6799 Nef/h Synthetic linker 8 6800-6807 Bovine growthhormone poly A 553 6808-7360 pUC derived 1473 7361-8833 Kanamycinresistance gene 623 8834-9456 pUC derived 334 9457-9790

B. Production of the Env DNA Plasmids

These DNA plasmids are designed to express HIV-1 Env glycoproteins thatare modified to reduce potential cellular toxicity by deletion of thefusion domain, the cleavage domains, and a portion of the interspace(1S) between heptad 1 (H1) and heptad 2 (H2).

a. VRC-5305 [pVR1012x/s CCR5-tropic gp145 Clade A (ΔCFI)/h]

The DNA plasmid, VRC-5305, is diagrammed in FIG. 33. The proteinsequence of the clade A Env polyprotein (gp160) from 92rw020(CCR5-tropic, GenBank accession number U08794) was used to create asynthetic version of the gene (clade A gp145ΔCFI/h) using codonsmodified for expression in human cells. The nucleotide sequence of theclade A CCR5-tropic gp145ΔCFI shows little homology to the 92rw020 gene,but the protein encoded is the same (note that GenBank U08794 sequencedoes contain the MR codons at the start of the sequence, so these wereinserted into the synthetic construct).

The truncated Env polyprotein contains the entire surface (SU) andtransmembrane (TM) proteins, but lacks the fusion and cytoplasmicdomains. Regions important for oligomer formation are retained,specifically the two helical coiled coil regions. The fusion andcleavage (F/CL) domains from amino acids 486-519 were deleted. The ISbetween H1 and H2 from amino acids 576-603 was also deleted. Theconstruct was then inserted into the pVR1012x/s backbone using XbaI andBamH1 restriction sites. The XbaI (17 nt upstream from ATG) to BamH1(1897 nt downstream from ATG) fragment contains a polylinker at the 5′end and the ATG was cloned into the XbaI to BamH1 sites of pVR1012x/sbackbone.

A summary of the predicted VRC-5305 domains is provided in Table 5. Theplasmid is 6836 nucleotide pairs (np) in length and has an approximatemolecular weight of 4.5 MDa. The sequence of VRC-5305 is provided as SEQID NO: 21.

TABLE 5 Summary of Predicted Domains of VRC-5305 Clade A CCR5-tropicgp145ΔCFI/h Fragment Name Predicted Fragment or Protein Domain FragmentSize (bp) Location pUC derived 247  1-247 CMV-IE enhancer/promoter 638248-885 CMV-IE 5′ UT region 244  886-1129 CMV IE intron 711 1130-1840Synthetic linker 82 1841-1922 CCR5-tropic gp145 Δ CFI/h 1881 1923-3803Synthetic linker 16 3804-3819 Bovine growth hormone 587 3820-4406 poly ApUC derived 1473 4407-5879 Kanamycin resistance gene 623 5880-6502 pUCderived 334 6503-6836

b. VRC-2805 [pVR1012x/s CCR5-tropic gp145(Δ F/CL Δ H IS)/h]

The DNA plasmid, VRC-2805, is diagrammed in FIG. 34. The proteinsequence of the clade B Env glycoprotein (gp160) from HXB2(CXCR4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (CXCR4gp160/h) using codons modified foroptimal expression in human cells. The nucleotide sequence X4gp160/hshows little homology to the HXB2 gene, but the protein encoded is thesame with the exception of the following amino acid substitutions: F53L,N94D, K192S, P470L, I580T, and Z653H. To produce a CCR5-tropic versionof the Env glycoprotein (R5gp160/h), the region encoding HIV-1 Envpolyprotein amino acids 205 to 361 from X4gp160/h (VRC-3300, describedin the WO 02/32943) was replaced with the corresponding region from theBaL strain of HIV-1 (GeneBank accession number M68893), again usinghuman preferred codons. The nucleotide sequence R5gp160/h shows littlehomology to the CCR5 gene, but the protein encoded is the same with thefollowing amino acid (aa) substitutions: I219N, L265V, N266T, and S268N.

The full-length CCR5-tropic version of the env gene from pR5gp160/h(VRC-3000, described in the WO 02/32943) was terminated after the codonfor amino acid 704. The truncated Env glycoprotein (gp145) contains theentire surface (SU) protein and a portion of the transmembrane (TM)protein including the fusion domain, the transmembrane domain, andregions important for oligomer formation, specifically, the two helicalcoiled coil motifs. The fusion and cleavage (F/CL) domains from aminoacids 503-536 were deleted. The IS between H1 and H2 from amino acids594-619 was also deleted. The construct was then inserted into thepVR1012x/s backbone using XbaI and BamH1 restriction sites. The XbaI (18nt upstream from ATG) to BamH1 (1937 nt downstream from ATG) fragmentthat contains a polylinker at the 5′ end and the ATG was cloned into theXbal to BamH1 sites of the 1012x/s backbone.

A summary of the predicted VRC-2805 domains is provided in Table 6. Theplasmid is 6869 nucleotide pairs (np) in length and has an approximatemolecular weight of 4.5 MDa. The kanamycin gene is incorporated into thebacterial vector backbone as a selectable marker. The sequence ofVRC-2805 is provided as SEQ ID NO: 22.

TABLE 6 Summary of Predicted Domains of Clade B VRC-2805 CCR5-tropicgp145ΔCFI/h Fragment Name Predicted Fragment or Protein Domain FragmentSize (bp) Location pUC derived 247  1-247 CMV-IE enhancer/promoter 638248-885 CMV-IE 5′ UT region 244  886-1129 CMV IE intron 711 1130-1840Synthetic linker 74 1841-1914 CCR5-tropic gp145 Δ CFI/h 1929 1915-3843Synthetic linker 9 3844-3852 Bovine growth hormone 587 3853-4439 poly ApUC derived 1473 4440-5912 Kanamycin resistance gene 623 5913-6535 pUCderived 334 6536-6869

c. VRC-5309 [pVR1012x/s CCR5-tropic gp145 Clade C (ΔCFI)/h]

The DNA plasmid, VRC-5309, is diagrammed in FIG. 35. The proteinsequence of the clade C Env polyprotein (gp145ΔCFI) from 97ZA012(CCR5-tropic, GenBank accession number AF286227) was used to create asynthetic version of the gene (clade C gp145ΔCFI/h) using codonsmodified for optimal expression in human cells. The nucleotide sequenceof the clade C CCR5-tropic gp145ΔCFI/h shows little homology to the gene97ZA012, but the protein encoded is the same except for the followingsubstitution: D605E. The truncated Env polyprotein contains the entireSU protein and the TM domain, but lacks the fusion domain andcytoplasmic domain. Regions important for oligomer formation areretained, specifically the two helical coiled coil motifs. The fusionand cleavage (F/CL) domains from amino acids 487-520 were deleted. Theinterspace between H1 and H2 from amino acids 577-604 was also deleted.The construct was then inserted into the pVR1012x/s backbone using XbaIand BamH1 restriction sites. The XbaI (17 nt upstream from ATG) to BamH1(1882 nt downstream from ATG) fragment contains a polylinker at the 5′end and the ATG was cloned into the XbaI to BamH1 sites of pVR1012x/sbackbone.

A summary of the predicted VRC-5309 domains is provided in Table 7. Theplasmid is 6829 nucleotide pairs (np) in length and has an approximatemolecular weight of 4.5 MDa. The kanamycin gene is incorporated into thebacterial vector backbone as a selectable marker. The sequence ofVRC-5309 is provided as SEQ ID NO: 23.

TABLE 7 Summary of Predicted Domains of VRC-5309 Clade C CCR5-tropicgp145ΔCFI/h Fragment Name Predicted Fragment or Protein Domain FragmentSize (bp) Location pUC derived 247  1-247 CMV-IE enhancer/promoter 638248-885 CMV-IE 5′ UT region 244  886-1129 CMV IE intron 711 1130-1840Synthetic linker 82 1841-1922 CCR5-tropic gp145 Δ CFI/h 1881 1923-3803Synthetic linker 9 3804-3812 Bovine growth hormone 587 3813-4399 poly ApUC derived 1473 4400-5872 Kanamycin resistance gene 623 5873-6495 pUCderived 334 6496-6829

C. Analysis of VRC-HIVDNA009-00-VP Plasmid Components Sequence Homologyto the Human Genome

Plasmids VRC-2805, 4306, 5305, and 5309 were sequenced by LarkTechnologies and the sequences subjected to a BLAST search using theBLASTN program searching the human est database. The search was doneusing parameters that only identified sequence homologies with expectedvalues (E values) of 0.01 or lower. This means that the statisticalpossibility of a homology occurring by chance alone is only 1/100.Anything at this level or lower (i.e. less than 1/100) will be picked upby the search. The results show numerous homologies at less than E=0.01.With one exception, the homologies are either in the pUC18 or the CMVpromoter portions of the plasmids. In addition, the sequences detectedwere between 85 and 100% identical to the sequences in the plasmids. Itis believed that these homologies are spurious and result fromcontamination of the human genome database with plasmid sequence fromcloning operations.

The other result shows homology with the bovine growth hormone poly Aterminator portion of the plasmid. The sequences detected were 90 to100% identical to the sequences in the plasmids. The human genesassociated with this homology were not related to human growth hormoneor related proteins. Upon further inspection, the description of theclones revealed that they had been excised from expression vectors(e.g., pDNA3) in which the cloning site was immediately adjacent to abovine growth hormone poly A terminator. As with the aforementionedpUC18 homologies, it is believed that these homologies are spurious andrepresent contamination of the database with plasmid sequence fromcloning operations.

D. Analytical Methods for the Drug Substance

In Vitro Transfection and Expression Assay. Expression testing for theindividual plasmids (gag-pol-nef, Clade A envelope, Clade B envelope andClade C envelope) is conducted prior to formulation of the vaccineproduct. Semi-quantitative values of the expression levels of theindividual plasmids is determined by comparing the intensity of thereactive protein bands on the Western Blot with the intensity ofstandards run under the same conditions. Once the plasmids are combined,expression is qualitatively verified using the same assay procedures.However, since the antibodies are cross-reactive, the level ofexpression of the specific clades of envelope in the mixture cannot bequantitated.

Expression of the Gag-Pol-Nef protein encoded by plasmid VRC-4306 isdetermined by quantitation of the level of Gag-Pol-Nef protein expressedby transfected HEK-293 (human embryonic kidney) cells. For transfection,10⁵ to 10⁶ cells are transfected with 1-5 μg of VRC-4306 plasmid DNAusing the calcium phosphate method. Cells are incubated for 14-20 hoursto allow for DNA uptake. Following a medium change, cells are grown foran additional 24-48 hours before harvesting. Transfection efficiency ismonitored using a human alkaline phosphatase vector in a similarbackbone.

After cell lysis, 10-50 μg of total cellular protein is loaded onto anSDS-PAGE gel to separate the crude lysate proteins. For quantitation,25-250 ng of HIV1 gag-β-gal fusion protein (Chemicon) is mixed with 10μg of cell lysate from non-transfected HEK293 cells and loaded onto thegel. Following electrophoresis for 1-3 hours, the proteins aretransferred to a nitrocellulose membrane (0.45 μm) for Western Blotanalysis. The membrane is blocked with skim milk to prevent non-specificbinding interaction prior to incubation with the primary antibody (mouseanti-HIV p24 [ICN Biomedical]) for 30-60 minutes. Following washing, themembrane is incubated for 30-60 minutes with HRP-sheep anti-mouse IgG.Visualization of the protein bands is achieved by incubating themembrane with chemiluminescent substrates and exposing to X-ray film for2-30 minutes. For quantitation, the intensity of the Gag-Pol-Nef proteinband is compared to the intensity of t of the HIV-1 gag-β-gal fusionprotein bands.

Analysis of Envelope protein expression by plasmids VRC-5305, VRC-2805,and VRC-5309 is determined in an analogous manner to that used foranalysis of VRC-4306. Following transfection with the plasmid, celllysate is harvested and analyzed by Western Blot analysis. Forimmunological detection, the membrane is incubated with human IgGantiserum against gp160 (NIH AIDS Research and Reference ReagentProgram). Protein expression levels are quantitated by comparing theintensity of the envelope protein bands to those of the purified gp160protein standard. Transfection efficiency is monitored using aβ-galactosidase expression vector in a similar backbone.

Part VII VRC-HIVDNA016-00-VP

Introduction

VRC-HIVDNA016-00-VP is a multi-plasmid DNA vaccine intended for use as apreventive vaccine for HIV-1. It is a mixture of six plasmids in equalconcentration. It was constructed to produce Gag, Pol, Nef and Env HIV-1proteins to potentially elicit broad immune responses to multiple HIV-1subtypes isolated in human infections.

Description of the Drug Substance

Name of the Drug Substance: VRC-4401 (SEQ ID NO: 24) Description: HIV-1Gag (Clade B) Molecular Weight: 3.9 MDa Nucleotide Base Pairs: 5886 Nameof the Drug Substance: VRC-4409 (SEQ ID NO: 25) Description: HIV-1 Pol(Clade B) Molecular Weight: 4.8 MDa Nucleotide Base Pairs: 7344 Name ofthe Drug Substance: VRC-4404 (SEQ ID NO: 26) Description: HIV-1 Nef(Clade B) Molecular Weight: 3.3 MDa Nucleotide Base Pairs: 5039 Name ofthe Drug Substance: VRC-5736 (SEQ ID NO: 27) Description: HIV-1 Env(Clade A) Molecular Weight: 4.2 MDa Nucleotide Base Pairs: 6305 Name ofthe Drug Substance: VRC-5737 (SEQ ID NO: 28) Description: HIV-1 Env(Clade B) Molecular Weight: 4.2 MDa Nucleotide Base Pairs: 6338 Name ofthe Drug Substance: VRC-5738 (SEQ ID NO: 29) Description: HIV-1 Env(Clade C) Molecular Weight: 4.2 MDa Nucleotide Base Pairs: 6298A. Construction of HIV-1 DNA Plasmids

The drug substances for VRC-HIVDNA016-00-VP are six closed circularplasmid DNA macromolecules (VRC-4401, VRC-4409, VRC-4404, VRC-5736, VRC5737, and VRC-5738) that have been produced in bacterial cell culturescontaining a kanamycin selection medium. In all cases, bacterial cellgrowth is dependent upon the cellular expression of the kanamycinresistance protein encoded by a portion of the plasmid DNA. Followinggrowth of bacterial cells harboring the plasmid, the plasmid DNA ispurified from cellular components.

Plasmids containing viral gene complementary DNAs (cDNAs) were used tosubclone the relevant inserts into plasmid DNA expression vectors thatuse the CMV/R promoter and the bovine growth hormone polyadenylationsequence. The HIV-1 gene inserts have been modified to optimizeexpression in human cells. The CMV/R promoter consists of translationalenhancer region of the CMV immediate early region 1 enhancer (CMV-IE)substituted with the 5′-untranslated HTLV-1 R-U5 region of the humanT-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) tooptimize gene expression further.

CMV/R-HIV-1 Clade B Gag/h (VRC-4401)

To construct DNA plasmid VRC-4401, diagrammed in FIG. 36, the proteinsequence of the gag polyprotein (Pr55, amino acids 1-432) from HXB2(GenBank accession number K03455) was used to create a synthetic versionof the gag gene using codons optimized for expression in human cells.The nucleotide sequence of the synthetic gag gene shows little homologyto the HXB2 gene, but the protein encoded is the same. The SalI/BamHIfragment of Gag (B) was excised from VRC 3900 (described in the WO02/32943), which contained the same insert in a pVR1012 backbone, andcloned into the SalI/BamHI sites of the CMV/R backbone described above.

A summary of predicted VRC-4401 domains is provided in Table 8. Theplasmid is 5886 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 3.9 MDa. The sequence of VRC-4401 isprovided as SEQ ID NO: 24.

TABLE 8 Summary of Predicted Domains of VRC-4401; HIV-1 Gag (Clade B)Fragment Name or Protein Domain Fragment Size (bp) Predicted FragmentpUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742 248-989HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 123 1221-1343Synthetic Linker 31 1344-1374 HIV-1 Gag (Clade B) 1509 1375-2883Synthetic Linker 23 2884-2906 Bovine Growth Hormone 548 2907-3454 Poly ApUC18 plasmid-derived 1311 3455-4765 Kanamycin Resistance Gene 8164766-5581 pUC18 plasmid-derived 305 5582-5886Construction of CMV/R Clade B Pol/h (VRC-4409)

To construct DNA plasmid VRC-4409 diagrammed in FIG. 37, the proteinsequence of the pol polyprotein (amino acids 3-1003) from NL4-3 (GenBankaccession number M19921) was used to create a synthetic version of thepol gene using codons optimized for expression in human cells. Toinitiate translation at the beginning of Pol, a methionine codon wasadded to the 5′-end of the synthetic polymerase gene to create the Pol/hgene. The Protease (PR) mutation is at amino acid 553 and is AGG->GGC oramino acids R->G. The Reverse Transcriptase (RT) mutation is at aminoacid 771 and is GAC->CAC or amino acids D->H. The Integrase (IN)mutation is at amino acid 1209 and is ACT->CAT or amino acids D->A. Thegene expressing Pol was inserted into the CMV/R backbone describedabove.

A summary of predicted VRC-4409 domains is provided in Table 9. Theplasmid is 7344 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 4.8 MDa. The sequence of VRC-4409 isprovided as SEQ ID NO: 25.

TABLE 9 Summary of Predicted Domains of VRC-4409; HIV-1 Pol (Clade B)Fragment Name or Protein Domain Fragment Size (bp) Predicted FragmentpUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742 248-989HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 123 1221-1343Synthetic Linker 5 1344-1348 HIV-1 Pol (Clade B) 3009 1349-4357 (Pr-,RT-, IN-) Synthetic Linker 7 4358-4364 Bovine Growth Hormone 5484365-4912 Poly A pUC18 plasmid-derived 1311 4913-6223 KanamycinResistance Gene 816 6224-7039 pUC18 plasmid-derived 305 7040-7344Construction of CMV/R HIV-1 Nef/h (VRC-4404)

To construct DNA plasmid VRC-4404, diagrammed in FIG. 38, the proteinsequence of the Nef protein from HIV-1 NY5/BRU (LAV-1) clone pNL4-3(GenBank accession number M19921) was used to create a synthetic versionof the Nef gene (Nef/h) using codons optimized for expression in humancells. The nucleotide sequence Nef/h shows little homology to the viralgene, but the protein encoded is the same. The Myristol site (GGC-Gly,amino acid 2-3) was deleted. The fragment encoding Nef was digested fromthe pVR1012 backbone in which it was originally inserted, withXbaI/BamHI, and then cloned into the XbaI/BamHI site of the CMV/Rbackbone described above.

A summary of predicted VRC-4404 domains is provided in Table 10. Theplasmid is 5039 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 3.3 MDa. The sequence of VRC-4404 isprovided as SEQ ID NO: 26.

TABLE 10 Summary of Predicted Domains of VRC-4404; HIV-1 Nef (Clade B)Fragment Name or Protein Domain Fragment Size (bp) Predicted FragmentpUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742 248-989HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 123 1221-1343Synthetic Linker 48 1344-1391 HIV-1 Nef (Clade B) 615 1392-2006 (DeltaMyr) Synthetic Linker 19 2007-2025 Bovine Growth Hormone 548 2026-2573Poly A pUC18 plasmid-derived 1345 2574-3918 Kanamycin Resistance Gene816 3919-4734 pUC18 plasmid-derived 305 4735-5039CMV/R-HIV-1 Clade A Env/h (VRC-5736)

To construct DNA plasmid VRC-5736, diagrammed in FIG. 39, the proteinsequence of the envelope polyprotein (gp160) from 92rw020 (R5-tropic,GenBank accession number U08794) was used to create a synthetic versionof the gene (Clade-A gp145ΔCFI) using codons altered for expression inhuman cells. Plasmids expressing the HIV-1 genes were made syntheticallywith sequences designed to disrupt viral RNA structures that limitprotein expression by using codons typically found in human cells. Thenucleotide sequence R5gp145ΔCFI shows little homology to the 92rw020gene, but the protein encoded is the same. The truncated envelopepolyprotein contains the entire SU protein and the TM domain, but lacksthe fusion domain and cytoplasmic domain. Regions important for oligomerformation may be partially functional. Heptad(H) 1, Heptad 2 and theirInterspace(IS) are required for oligomerization. The Fusion and Cleavage(F/CL) domains, from amino acids 486-519, have been deleted. TheInterspace (IS) between Heptad (H) 1 and 2, from amino acids 576-604,have been deleted. The XbaI (18 nt up-stream from ATG) to BamH1 (1912 ntdown-stream from ATG) fragment which contains polylinker at the 5′ end,Kozak sequence and ATG was cloned into the XbaI to BamH1 sites of theCMV/R backbone described above.

EnvA summary of predicted VRC-5736 domains is provided in Table 11. Theplasmid is 6305 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 4.2 MDa. The sequence of VRC-5736 isprovided as SEQ ID NO: 27.

TABLE 11 Summary of Predicted Domains of VRC-5736; HIV-1 Env (Clade A)Fragment Name or Protein Domain Fragment Size (bp) Predicted FragmentpUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742 248-989HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 123 1221-1343Synthetic Linker 48 1344-1391 HIV-1 Env (Clade A), gp145 1881 1392-3272(ΔCFI)/h Synthetic Linker 19 3273-3291 Bovine Growth Hormone 5483292-3839 Poly A pUC18 plasmid-derived 1345 3840-5184 KanamycinResistance Gene 816 5185-6000 pUC18 plasmid-derived 305 6001-6305Construction of CMV/R Clade B Env/h (VRC-5737)

To construct DNA plasmid VRC-5737 diagrammed in FIG. 40, the proteinsequence of the envelope polyprotein (gp160) from HXB2 (X4-tropic,GenBank accession number K03455) was used to create a synthetic versionof the gene (X4gp160/h) using codons optimized for expression in humancells. The nucleotide sequence X4gp160/h shows little homology to theHXB2 gene, but the protein encoded is the same with the following aminoacid substitutions: F53L, N94D, K192S, 1215N, A224T, A346D, and P470L.To produce an R5-tropic version of the envelope protein (R5gp160/h), theregion encoding HIV-1 envelope polyprotein amino acids 275 to 361 fromX4gp160/h (VRC3300) were replaced with the corresponding region from theBaL strain of HIV-1 (GeneBank accession number M68893, again using humanpreferred codons). The full-length R5-tropic version of the envelopeprotein gene from pR5gp160/h (VRC3000, described in the WO 02/32943) wasterminated after the codon for amino acid 704. The truncated envelopepolyprotein (gp145) contains the entire SU protein and a portion of theTM protein including the fusion domain, the transmembrane domain, andregions important for oligomer formation. Heptad(H) 1, Heptad 2 andtheir Interspace (IS) are required for oligomerization. The Fusion andCleavage (F/CL) domains, from amino acids 503-536, have been deleted.The Interspace (IS) between Heptad (H) 1 and 2, from amino acids593-620, have been deleted. The expression vector backbone is CMV/R,described above.

A summary of predicted VRC-5737 domains is provided in Table 12. Theplasmid is 6338 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 4.2 MDa. The sequence of VRC-5737 isprovided as SEQ ID NO: 28.

TABLE 12 Summary of Predicted Domains of VRC-5737; HIV-1 Env (Clade B)Fragment Name or Protein Domain Fragment Size (bp) Predicted FragmentpUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742 248-989HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 123 1221-1343Synthetic Linker 40 1344-1383 HIV-1 Env (Clade B), gp145 1929 1384-3312(ΔCFI)/h Synthetic Linker 12 3313-3324 Bovine Growth Hormone 5483325-3872 Poly A pUC18 plasmid-derived 1345 3873-5217 KanamycinResistance Gene 816 5218-6033 pUC18 plasmid-derived 305 6034-6338Construction of CMV/R Clade C Env/h (VRC-5738)

To construct DNA plasmid VRC-5738, diagrammed in FIG. 41, the proteinsequence of the envelope polyprotein (gp145ΔCFI) from 97ZA012(R5-tropic, GenBank accession number AF286227) was used to create asynthetic version of the gene (Clade-C gp145ΔCFI) using codons optimizedfor expression in human cells. The nucleotide sequence R5gp145ΔCFI showslittle homology to the gene 97ZA012, but the protein encoded is thesame. The truncated envelope polyprotein contains the entire SU proteinand the TM domain, but lacks the fusion domain and cytoplasmic domain.Regions important for oligomer formation may be partially functional.Heptad(H) 1, Heptad 2 and their Interspace(IS) are required foroligomerization. The Fusion and Cleavage (F/CL) domains, from aminoacids 487-520, have been deleted. The Interspace (IS) between Heptad (H)1 and 2, from amino acids 577-605, have been deleted. The XbaI (18 ntup-stream from ATG) to BamH1 (1914 nt down-stream from ATG) fragmentwhich contains polylinker at the 5′ end, Kozak sequence and ATG wascloned into the XbaI to BamH1 sites of the CMV/R backbone.

A summary of predicted VRC-5738 domains is provided in Table 13. Theplasmid is 6298 nucleotide base pairs (bp) in length and has anapproximate molecular weight of 4.2 MDa. The sequence of VRC-5738 isprovided as SEQ ID NO: 29.

TABLE 13 Summary Table of Predicted Domains of VRC-5738; HIV-1 Env(Clade C) Fragment Name or Protein Domain Fragment Size (bp) PredictedFragment pUC18 plasmid-derived 247  1-247 CMV-IE Enhancer/Promoter 742248-989 HTLV-1 R region 231  990-1220 CMV IE Splicing Acceptor 1231221-1343 Synthetic Linker 48 1344-1391 HIV-1 Env (Clade C), gp145 18811392-3272 (ΔCFI)/h Synthetic Linker 12 3273-3284 Bovine Growth Hormone548 3285-3832 Poly A pUC18 plasmid-derived 1345 3833-5177 KanamycinResistance Gene 816 5178-5993 pUC18 plasmid-derived 305 5994-6298B. Analysis of HIV-1 Plasmid Sequence Homology to the Human Genome

VRC-4401, 4409, 4404, 5736, 5737 and 5738 plasmids were sequenced byLark Technologies and the sequences subjected to a BLAST search of thehuman genome database. The search was done using parameters which onlyidentified sequence homologies with expected values (E values) of 0.01or lower. This means that the statistical possibility of a homologyoccurring by chance alone is only 1/100. Anything at this level or lower(i.e. less than 1/100) will be picked up by the search.

C. Analytical Methods for the Drug Substance

In Vitro Transfection and Expression Assay. Expression testing for theindividual plasmids and the final formulated drug product will beconducted prior to release of the vaccine product. Qualitativeexpression of the plasmid proteins is verified by comparing the reactiveprotein bands on the Western blot with the standards run under the sameconditions. Once the plasmids are combined, expression will be verifiedusing the same assay procedures. Expression is determined by detectingproteins expressed by transfected 293 human embryonic kidney (HEK)cells. For transfection, 10⁵ to 10⁶ cells are transfected with 1-5 μg ofplasmid DNA using the calcium phosphate method. Cells are incubated for14-20 hours to allow for DNA uptake. Following a medium change, cellsare grown for an additional 24-48 hours before harvesting. Transfectionefficiency is monitored using a known similar vector in the samebackbone. After cell lysis, 10 μg of an appropriate amount of totalcellular protein is loaded onto an SDS-PAGE gel to separate the crudelysate proteins.

Following electrophoresis for approximately 1.5 hours, the proteins aretransferred to a nitrocellulose membrane (0.45 μm) for Western blotanalysis. The membrane is blocked with skim milk to prevent non-specificbinding interaction prior to incubation with the primary antibody for 60minutes. Following washing, the membrane is incubated for 45 minuteswith HRP conjugated second antibody. Visualization of the protein bandsis achieved by incubating the membrane with chemiluminescent substratesand exposing to X-ray film for 2 minutes or an appropriate time.Expression of protein produced by transfected cells is determined byobserving the intensity of expressed protein on the Western blot. Theassay is being further developed to allow for semi-quantitative analysisof protein expression by the vaccine plasmids.

Part VIII VRC NIH ADV014-00-VP

Description of the Study Agent VRC-HIVADV014-00-VP

The recombinant adenoviral vector product VRC-HIVADV014-00-VP (rAd) is areplication-deficient, combination vaccine containing four recombinantserotype 5 adenoviral vectors. These vectors contain gene sequences thatcode for Clade B HIV-1 Gag and Pol as well as Clade A, Clade B, andClade C Env protein. In vivo expression by these vectors producesimmunogens that induce an immune response against HIV. The envelopegenes were chosen as representative primary isolates from each of thethree clades.

The process for constructing the four VRC-HIVADV014-00-VP recombinantadenoviral vectors is based upon a rapid vector construction system(AdFAST™, GenVec, Inc.) used to generate adenoviral vectors that expressthe four HIV antigens gp140(A), gp140(B)dv12, gp140(C) and GagPol(B)driven by the cytomegalovirus (CMV) immediate-early promoter.Manufacturing is based upon production in a 293-ORF6 cell line (Brough,D. E. at al. 1996 J Virol 70:6497-6501), yielding adenoviral vectorsthat are replication deficient. The vectors are purified using CsClcentrifugation. The product is formulated as a sterile liquid injectabledosage form for intramuscular injection.

1. Production of the gag-pol Adenoviral Vector

AdtGagPol(B).11D (SEQ ID NO: 33)

The protein sequences of the Gag and Pol proteins from an HIV-1 Clade Bwere used to create a synthetic polyprotein version of the gag-pol genesusing codons optimized for expression in human cells. The synthetic gaggene is from HIV-1 Clade B strain HXB2 (GenBank accession numberK03455), and the synthetic poi gene (pol/h) is from HIV-1 Clade B NL4-3(GenBank accession number M19921). The pol gene is nonfunctional becauseit is present as a fusion protein. Mutations were introduced in thesynthetic protease and reverse transcriptase genes. The proteasemodification prevents processing of the pol gene product, and reducesthe potential for functional protease, reverse transcriptase andintegrase enzymatic activity. The cDNA used to produce AdtGagPol(B).11Dis similar to an HIV-1 DNA vaccine VRC-4302 (described in WO 02/32943)which was tested and shown to have no reverse transcriptase activity. Nomodifications were made to the gag. To construct the adenoviral vector,the HIV-1 DNA sequence was subcloned using standard recombinant DNAtechniques into an expression cassette in an E1-shuttle plasmid.

2. Production of the env Adenoviral Vectors

Adgp140(A).11D (SEQ ID NO: 30)

The protein sequence of the envelope polyprotein (gp160) from 92rw020(CCR5-tropic, GenBank accession number U08794) was used to create asynthetic version of the gene (Clade-A gp140ΔCFI) using codons alteredfor expression in human cells. Plasmids expressing the HIV-1 genes weremade synthetically with sequences designed to disrupt viral RNAstructures that limit protein expression by using codons typically foundin human cells. To construct the adenoviral vector, the HIV-1 DNAsequence was subcloned using standard recombinant DNA techniques into anexpression cassette in an E1-shuttle plasmid.

Adtgp140dv12(B).11D (SEQ ID NO: 32)

The protein sequence of the envelope polyprotein (gp160) from HXB2(X4-tropic, GenBank accession number K03455) was used to create asynthetic version of the gene (X4gp160/h) using codons optimized forexpression in human cells. To produce an CCR5-tropic version of theenvelope protein (R5gp160/h), the region encoding HIV-1 envelopepolyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) werereplaced with the corresponding region from the BaL strain of HIV-1(GenBank accession number M68893, again using human preferred codons).The full-length CCR5-tropic version of the envelope protein gene frompR5gp160/h (VRC3000) was terminated after the codon for amino acid 680.The truncated Env glycoprotein (gp140) contains the entire surfaceprotein and the ectodomain of gp41 including the fusion domain, andregions important for oligomer formation, specifically two helicalcoiled coil motifs. The Env V1 and V2 loops were deleted to improve thestability and yield of the vector in the producer cell line. Twoadditional amino acids were incorporated immediately after the deletiondue to creation of a restriction enzyme site. In order to construct theadenoviral vector, the HIV-1 DNA sequence was subcloned using standardrecombinant DNA techniques into an expression cassette in an E1-shuttleplasmid.

Adgp140(C).11D (SEQ ID NO: 31)

The protein sequence of the envelope polyprotein (gp140ΔCFI) from97ZA012 (CCR5-tropic, GenBank accession number AF286227) was used tocreate a synthetic version of the gene (Clade-C gp140ΔCFI) using codonsoptimized for expression in human cells. To construct the adenoviralvector, the HIV-1 DNA sequence was subcloned using standard recombinantDNA techniques into an expression cassette in an E1-shuttle plasmid.

All Four Adenoviral Vectors

The four E1-shuttle plasmid was recombined in Escherichia coli (E. coli)BjDE3 bacteria with the GV11 adenovector based AdFAST™ plasmidpAdE1(BN)E3(10)E4(T1S1) to generate the adenoviral vector plasmids. Thereplication-deficient adenoviral vectors AdtGagPol(B).11D,Adgp140(A).11D, Adtgp140dv12(B).11D, and Adgp140(C).11D were thengenerated by introducing the adenoviral vector plasmid into thepackaging cell line, 293-ORF6.

Part IX Clinical Data

Preliminary immunogenicity data through Week 12 from the clinical study(VRC-004) of VRC-HIVDNA-009-00-VP vaccine, when sorted by treatmentassignment indicate that CD4⁺ responses were detected in nearly 100% ofrecipients at all dose levels. CD8⁺ responses were detected in nearlyhalf. The greatest responses (in frequency and magnitude) were generallyobserved as directed against Env. Greater responses were observed in the4 mg and 8 mg dose compared to the 2 mg dose, although not statisticallysignificant given the small number of subjects at the 2 mg dose. Alarger response was observed after 3 injections compared to 2 injectionsat both the 4 mg and 8 mg dose levels, although it was not statisticallysignificant and there is no way to determine if this was due to the 3rdinjection or simply a maturation of the response following the 2ndinjection. Definitive cellular immune responses were first detectablewith the 4 mg and 8 mg dose at the 6-week time point (2 weeks after thesecond injection).

Serological responses to immunizations were analyzed by ELISA andWestern Blot. None of the 2 mg dose subjects showed evidence of humoralimmunity by standard HIV ELISA or Western blot. HIV ELISA responses weredetected in 11 of the 20 (55%) subjects vaccinated with the 4 mg dose,and 3 of 15 (20%) subjects vaccinated with the 8 mg dose. The studysubjects with vaccine-induced antibody had indeterminate or negativeWestern blots. With the schedule of evaluations used in VRC 004, studyweek 8 is the earliest timepoint at which a positive vaccine-induced HIVELISA was detected, although more often a positive ELISA was firstdetected at study week 12 and some have been first detected at latertimepoints. It appears that over time the strength of thevaccine-induced HIV ELISA reaction diminishes as indicated by decreasingoptical density (O.D.) measurements reported for sequential ELISAmeasurements.

Part X Additional Constructs

TABLE 14 V3 1AB modified envelope constructs. FIG. SEQ VRC NO: ConstructNO: ID NO: VRC 5747 CMV/R-Clade B gp145(ΔCFI)(ΔV12)(V3-1AB-clade C- 4638 SA)/h VRC 5753 CMV/R-gp145(ΔCFI)(ΔV1-2)(ΔV3)(1AB)(Clade A) 47 39 VRC5754 CMV/R-gp145(ΔCFI)(ΔV1-2)(ΔV3)(1AB)(Clade SA-C) 48 40 VRC 5755pAdApt LoxP CMV TbGH(+) 49 41 gp140ΔCFIΔV1V2(1AB)(Bal)/h VRC 5766 pAdAptLoxP CMV TbGH(+) gp140(ΔCFI)(V3-1AB) 50 42 Clade A/h VRC 5767 pAdAptLoxP CMV TbGH(+) gp140(ΔCFI)(ΔV12)(V3- 51 43 1AB)h Clade A VRC 5768pAdApt LoxP CMV TbGH(+) gp140(ΔCFI)(ΔV1-2) Clade 52 44 B(V3-1AB-cladeA)/h VRC 5769 pAdApt LoxP CMV TbGH(+) gp140(ΔCFI)(V3-1AB) 53 45 CladeC(SA)/h VRC 5770 pAdApt LoxP CMV TbGH(+) gp140(ΔCFI)(ΔV1-2)(V3- 54 461AB)h Clade C(SA) VRC 5771 CMV/R gp145(ΔCFI)(V3-1AB)/h Clade A 55 47 VRC5772 CMV/R gp145(ΔCFI)(ΔV1-2)Clade B (V3-1AB-cladeA)/h 56 48 VRC 5773CMV/R gp145(ΔCFI)(V3-1AB)/h Clade C(SA) 57 49

TABLE 15 Deletions and mutations in V1V2 region on Bal gp145ΔCFI(V3-1AB)backbone FIG. SEQ ID Construct NO: NO:CMVR-gp145ΔCFIΔV1(V2ΔLR)(V3-1AB)(Bal) 58 50CMVR-gp145ΔCFI(V12ΔG)(V3-1AB)(Bal) 59 51CMVR-gp145ΔCFI(V1ΔG)(V2ΔLR)(V3-1AB)(Bal) 60 52CMVR-gp145ΔCFI(V1ΔG)(V2ΔM)(V3-1AB)(Bal) 61 53CMVR-gp145ΔCFI(V1ΔG)ΔV2(V3-1AB)(Bal) 62 54CMVR-gp145ΔCFI(V1ΔLR)(V2ΔG)(V3-1AB)(Bal) 63 55CMVR-gp145ΔCFI(V1ΔLR)ΔV2(V3-1AB)(Bal) 64 56CMVR-gp145ΔCFI(V1ΔM)(V2ΔG)(V3-1AB)(Bal) 65 57CMVR-gp145ΔCFI(V1ΔM)ΔV2(V3-1AB)(Bal) 66 58 CMVR-gp145ΔCFI(V3-1AB)(Bal)67 59 CMVR-gp145ΔCFIΔV1(V2ΔG)(V3-1AB)(Bal) 68 60CMVR-gp145ΔCFIΔV1(V2ΔM)(V3-1AB)(Bal) 69 61CMVR-gp145ΔCFIΔV1(V3-1AB)(Bal) 70 62 CMVR-gp145ΔCFIΔV1V2(V3-1AB)(Bal) 7163 CMVR-gp145ΔCFIΔV2(V3-1AB)(Bal) 72 64

TABLE 16 Chimeric constructs. FIG. SEQ ID VRC NO: Construct NO: NO: VRC5781 pAdApt LoxP CMV TbGH(+) Bal-gp140ΔCFI(C1-V2-CSA)h 73 65 VRC 5782CMVR-Bal-gp145ΔCFI(C1-V2 clade C-SA)(CBBB) 74 66 VRC 5783 pAdApt LoxPCMV TbGH(+) Bal-gp140ΔCFI(C2-C3- 75 67 CSA)h(BCBB) VRC 5784CMVR-Bal-gp145ΔCFI(C2-C3-CSA)h(BCBB) 76 68 VRC 5785 pAdApt LoxP CMVTbGH(+) Bal-gp140ΔCFI(V4-C5- 77 69 CSA)/h(BBCB) VRC 5786CMVR-Bal-gp145ΔCFI(V4-C5-CSA)/h(BBCB) 78 70 VRC 5787 pAdApt LoxP CMVTbGH(+) 79 71 gp140ΔCFIM383(Bal)/h(BBBB) VRC 5788CMVR-Bal-gp145ΔCFIM383(Bal)(BBBB) 80 72 VRC 5789 pAdApt LoxP CMV TbGH(+)C(SA)gp140ΔCFI(C1-V2 81 73 Bal)/h(BCCC) VRC 5790CMVR-C(SA)gp145ΔCFI(C1-V2 clade B-Bal)(BCCC) 82 74 VRC 5791 pAdApt LoxPCMV TbGH(+) C(SA)gp140ΔCFI(C2-C3 83 75 Bal)/h(CBCC) VRC 5792CMVR-C(SA)gp145ΔCFI(C2-C3 clade B-Bal)(CBCC) 84 76 VRC 5793 pAdApt LoxPCMV TbGH(+) C(SA)gp140ΔCFI(V4-C5 85 77 Bal)/h(CCBC) VRC 5794CMVR-C(SA)gp145ΔCFI(V4-C5 clade B-Bal)/h(CCBC) 86 78 CMVR gp145 ΔCFI(CCCC) — 79

1. A composition comprising: (i) a plasmid comprising SEQ ID NO: 24,(ii) a plasmid comprising SEQ ID NO: 25, (iii) a plasmid comprising SEQID NO: 26, (iv) a plasmid comprising SEQ ID NO: 27, (v) a plasmidcomprising SEQ ID NO: 28, and (vi) a plasmid comprising SEQ ID NO: 29,and a pharmaceutically acceptable carrier.
 2. A composition designatedVRC-HIVDNA016-00-VP.
 3. A method of inducing an immune response againstHIV-1 in an animal, which method comprises administering the compositionof claim 1 to an animal, whereupon an immune response against HIV-1 isinduced in the animal.